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1.
PLoS One ; 13(10): e0205888, 2018.
Article in English | MEDLINE | ID: mdl-30335858

ABSTRACT

Adipose tissue, which is conserved in higher eukaryotes, plays central roles in controlling the body's energy balance, including excess energy storage and energy expenditure during starvation. In adipogenesis, intranuclear receptor, peroxisome proliferator-activated receptor gamma (PPARγ) is a key molecule, and PPARγ agonists can promote adipogenesis. Many studies on the in vitro screening of PPARγ agonists with compounds derived from various materials have been reported; however, in vivo assays for quick examination of these feeding effects have not been established. In this study, we developed a technique using a lipophilic fluorescent reagent, Nile red to quantitatively estimate the adipose tissue volumes by using Japanese rice fish, medaka (Oryzias latipes) and studied effects of dietary soy sauce oil (SSO), which is a discarded by-product from Japanese traditional food and is known to have PPARγ-agonistic activity, on adipogenesis. We found that SSO feeding increased the adipose tissue volumes, and the expression levels of adipogenesis-related genes increased in these medaka larvae. These results suggest that SSO feeding increases the adipose tissue volumes through adipogenesis promotion by PPARγ-agonistic activity in medaka, and medaka is a powerful model for studying adipogenesis. Furthermore, our study also demonstrates the availability of SSO as a dietary additive for farmed fish.


Subject(s)
Adipogenesis/drug effects , Adipose Tissue/drug effects , Animal Feed/analysis , Dietary Fats, Unsaturated/administration & dosage , Larva/drug effects , Oryzias/genetics , PPAR gamma/genetics , Activin Receptors, Type I/genetics , Activin Receptors, Type I/metabolism , Adipogenesis/genetics , Adiponectin/genetics , Adiponectin/metabolism , Adipose Tissue/metabolism , Animals , Animals, Genetically Modified , Aquaculture , Diet/methods , Energy Metabolism/drug effects , Energy Metabolism/genetics , Fatty Acid-Binding Proteins/genetics , Fatty Acid-Binding Proteins/metabolism , Fish Proteins/genetics , Fish Proteins/metabolism , Fluorescent Dyes/administration & dosage , Fluorescent Dyes/chemistry , Gene Expression Regulation , Genes, Reporter , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Larva/genetics , Larva/growth & development , Larva/metabolism , Lipid Metabolism/drug effects , Lipid Metabolism/genetics , Oryzias/growth & development , Oryzias/metabolism , Oxazines/administration & dosage , Oxazines/chemistry , PPAR gamma/agonists , PPAR gamma/metabolism , Soy Foods/analysis
3.
Chromosome Res ; 23(4): 649-62, 2015 Dec.
Article in English | MEDLINE | ID: mdl-25947045

ABSTRACT

We have developed a convenient multicolor fluorescent in situ hybridization (FISH) (five-, four-, three-, and two-color FISHs) for detecting specific genes/DNA segments on the human chromosomes. As a foundation of multicolor FISH, we first isolated 80 bacterial artificial chromosome (BAC) probes that specifically detect the peri-centromeres (peri-CEN) and subtelomeres (subTEL) of 24 different human chromosomes (nos. 1~22, X, and Y) by screening our homemade BAC library (Keio BAC library) consisting of 200,000 clones. Five-color FISH was performed using human DNA segments specific for peri-CEN or subTEL, which were labeled with five different fluorescent dyes [7-diethylaminocoumarin (DEAC): blue, fluorescein isothiocyanate (FITC): green, rhodamine 6G (R6G): yellow, TexRed: red, and cyanine5 (Cy5): purple]. To observe FISH signals under a fluorescence microscope, five optic filters were carefully chosen to avoid overlapping fluorescence emission. Five-color FISH and four-color FISH enabled us to accurately examine the numerical anomaly of human chromosomes. Three-color FISH using two specific BAC clones, that distinguish 5' half of oncogene epidermal growth factor receptor (EGFR) from its 3' half, revealed the amplification and truncation of EGFR in EGFR-overproducing cancer cells. Moreover, two-color FISH readily detected a fusion gene in leukemia cells such as breakpoint cluster region (BCR)/Abelson murine leukemia viral oncogene homologue (ABL) on the Philadelphia (Ph') chromosome with interchromosomal translocation. Some other successful cases such as trisomy 21 of Down syndrome are presented. Potential applications of multicolor FISH will be discussed.


Subject(s)
Chromosomes, Human , In Situ Hybridization, Fluorescence/methods , Chromosome Aberrations , Chromosome Disorders/diagnosis , Chromosome Disorders/genetics , Chromosomes, Artificial, Bacterial , DNA Probes , Genomic Library , Humans , Microscopy, Fluorescence, Multiphoton , Staining and Labeling
4.
Hum Mutat ; 36(8): E2430-40, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25907632

ABSTRACT

We previously isolated PARKIN (PARK2) as a gene responsible for a unique sort of Parkinson disease, namely Autosomal Recessive Juvenile Parkinsonism (ARJP). In this study, we surveyed all the available literature describing PARK2 gene/Parkin protein mutations found in Parkinson disease patients. Only carefully evaluated data were deposited in the graphical database MutationView (http://mutview.dmb.med.keio.ac.jp) to construct KM-parkin-DB, an independent sub-set database. Forty-four articles were selected for data curation regarding clinical information such as ethnic origins, manifested symptoms, onset age, and hereditary patterns as well as mutation details including base changes and zygosity. A total of 366 cases were collected from 39 ethnic origins and 96 pathogenic mutations were found. PARK2 gene mutations were found also in some general Parkinson disease patients. The majority (63%) of mutations in PARK2 were restricted to two particular domains (UBL and RING1) of the Parkin protein. In these domains, two major mutations, a large deletion (DelEx3) and a point mutation (p.Arg275Trp), were located.


Subject(s)
Mutation , Parkinsonian Disorders/genetics , Ubiquitin-Protein Ligases/genetics , Databases, Nucleic Acid , Humans , Protein Isoforms/chemistry , Protein Isoforms/genetics , Protein Structure, Tertiary , Ubiquitin-Protein Ligases/chemistry
5.
Nature ; 521(7551): 217-221, 2015 May 14.
Article in English | MEDLINE | ID: mdl-25778702

ABSTRACT

Vertebrates have a unique 3D body shape in which correct tissue and organ shape and alignment are essential for function. For example, vision requires the lens to be centred in the eye cup which must in turn be correctly positioned in the head. Tissue morphogenesis depends on force generation, force transmission through the tissue, and response of tissues and extracellular matrix to force. Although a century ago D'Arcy Thompson postulated that terrestrial animal body shapes are conditioned by gravity, there has been no animal model directly demonstrating how the aforementioned mechano-morphogenetic processes are coordinated to generate a body shape that withstands gravity. Here we report a unique medaka fish (Oryzias latipes) mutant, hirame (hir), which is sensitive to deformation by gravity. hir embryos display a markedly flattened body caused by mutation of YAP, a nuclear executor of Hippo signalling that regulates organ size. We show that actomyosin-mediated tissue tension is reduced in hir embryos, leading to tissue flattening and tissue misalignment, both of which contribute to body flattening. By analysing YAP function in 3D spheroids of human cells, we identify the Rho GTPase activating protein ARHGAP18 as an effector of YAP in controlling tissue tension. Together, these findings reveal a previously unrecognised function of YAP in regulating tissue shape and alignment required for proper 3D body shape. Understanding this morphogenetic function of YAP could facilitate the use of embryonic stem cells to generate complex organs requiring correct alignment of multiple tissues.


Subject(s)
Body Size/genetics , Fish Proteins/metabolism , Morphogenesis/genetics , Oryzias/anatomy & histology , Oryzias/embryology , Actomyosin/metabolism , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/metabolism , Animals , Embryo, Nonmammalian/anatomy & histology , Embryo, Nonmammalian/embryology , Embryo, Nonmammalian/metabolism , Fish Proteins/genetics , GTPase-Activating Proteins/metabolism , Genes, Essential/genetics , Gravitation , Humans , Mutation/genetics , Organ Size/genetics , Oryzias/genetics , Phenotype , Protein Serine-Threonine Kinases/genetics , Protein Serine-Threonine Kinases/metabolism , Signal Transduction , Spheroids, Cellular/cytology , Spheroids, Cellular/metabolism
6.
Biol Open ; 3(11): 1071-82, 2014 Oct 17.
Article in English | MEDLINE | ID: mdl-25326516

ABSTRACT

We previously established three mouse cell lines (Aire(+)TEC1, Aire(+)TEC2 and Aire(+)DC) from the medullary thymic epithelial cells (mTECs) and dendritic cells (mDCs). These cells constitutively expressed "autoimmune regulator (Aire) gene" and they exhibited various features of self antigen-presenting cells (self-APCs) present in the thymic medullary region. Here, we confirmed our previous observation that Aire(+) thymic epithelial cells adhere to fresh thymocytes and kill them by inducing apoptosis, thus potentially reproducing in vitro some aspects of the negative selection of T cells in vivo. In this system, a single Aire(+) cell appeared able to kill ∼30 thymocytes within 24 hrs. Moreover, we observed that ectopic expression of peripheral tissue-specific antigens (TSAs), and expression of several surface markers involved in mTEC development, increased as Aire(+) cell density increases toward confluency. Thus, these Aire(+) cells appear to behave like differentiating mTECs as if they pass through the developmental stages from intermediate state toward mature state. Surprisingly, an in vitro co-culture system consisting of Aire(+) cells and fractionated sub-populations of fresh thymocytes implied the possible existence of two distinct subtypes of thymocytes (named as CD4(+) killer and CD4(-) rescuer) that may determine the fate (dead or alive) of the differentiating Aire(+)mTECs. Thus, our in vitro co-culture system appears to mimic a part of "in vivo thymic crosstalk".

7.
Biosci Biotechnol Biochem ; 78(7): 1123-8, 2014.
Article in English | MEDLINE | ID: mdl-25229845

ABSTRACT

Resveratrol was glucosylated to its 3- and 4'-ß-glucosides by cultured cells of Phytolacca americana. On the other hand, cultured P. americana cells glucosylated pterostilbene to its 4'-ß-glucoside. P. americana cells converted piceatannol into its 4'-ß-glucoside. The 3- and 4'-ß-glucosides of resveratrol were further glucosylated to 3- and 4'-ß-maltosides of resveratrol, 4'-ß-maltoside of which is a new compound, by cyclodextrin glucanotransferase. Resveratrol 3-ß-glucoside and 3-ß-maltoside showed low 2,2-diphenyl-1-picrylhydrazyl free-radical-scavenging activity, whereas other glucosides had no radical-scavenging activity. Piceatannol 4'-ß-glucoside showed the strongest inhibitory activity among the stilbene glycosides towards histamine release from rat peritoneal mast cells. Pterostilbene 4'-ß-glucoside showed high phosphodiesterase inhibitory activity.


Subject(s)
Glycosides/chemistry , Stilbenes/chemical synthesis , Stilbenes/pharmacology , Animals , Anti-Allergic Agents/chemical synthesis , Anti-Allergic Agents/chemistry , Anti-Allergic Agents/pharmacology , Biphenyl Compounds , Cell Line , Chemistry Techniques, Synthetic , Free Radical Scavengers/chemical synthesis , Free Radical Scavengers/chemistry , Free Radical Scavengers/pharmacology , Histamine Release/drug effects , Male , Neuroprotective Agents/chemical synthesis , Neuroprotective Agents/chemistry , Neuroprotective Agents/pharmacology , Phosphodiesterase Inhibitors/chemical synthesis , Phosphodiesterase Inhibitors/chemistry , Phosphodiesterase Inhibitors/pharmacology , Picrates , Rats , Resveratrol , Stilbenes/chemistry
8.
PLoS One ; 9(8): e105904, 2014.
Article in English | MEDLINE | ID: mdl-25157574

ABSTRACT

Commensal bacteria in gastrointestinal tracts are reported to function as an environmental factor to regulate intestinal inflammation and immune responses. However, it remains largely unknown whether such bacterial function exerts any effect on other immune organs distant from the intestine. In this study, the influence of commensal bacteria in the thymus, where T cell lineages develop into mature type to form proper repertoires, was investigated using germ-free (GF) mice and Nod1-deficient mice lacking an intracellular recognition receptor for certain bacterial components, in which a commensal bacterial effect is predicted to be less. In both mice, there was no significant difference in the numbers and subset ratios of thymocytes. Interestingly, however, autoimmune regulator (Aire) expression in thymic epithelial cells (TECs), main components of the thymic microenvironment, was decreased in comparison to specific pathogen-free (SPF) mice and Nod1 wild-type (WT) mice, respectively. In vitro analysis using a fetal thymus organ culture (FTOC) system showed that Aire expression in TECs was increased in the presence of a bacterial component or a bacterial product. These results suggest that through their products, commensal bacteria have the potential to have some effect on epithelial cells of the thymus in tissues distant from the intestine where they are originally harbored.


Subject(s)
Microbiota/physiology , Thymus Gland/metabolism , Transcription Factors/metabolism , Animals , Gastrointestinal Tract/microbiology , Gene Expression , Germ-Free Life , Mice, Inbred BALB C , Mice, Knockout , Thymocytes/metabolism , Thymus Gland/cytology , Tissue Culture Techniques , Transcription Factors/genetics , Transcriptional Activation , AIRE Protein
9.
J Dermatol Sci ; 76(1): 10-5, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25086748

ABSTRACT

BACKGROUND: Filaggrin (FLG) is a major protein component of the stratum corneum (SC) layer, and FLG loss-of-function mutations are a predisposing factor for atopic dermatitis (AD). Previous cohort studies of children from northern and western Europe have reported FLG loss-of-function mutation frequencies of 15.1-20.9% and 5.8-13.0% in AD and non-AD groups, respectively. OBJECTIVE: To elucidate the association between AD prevalence of FLG loss-of-function mutation carriers and climate conditions, we determined the AD prevalence and FLG loss-of-function mutation frequencies in a cohort of children from Ishigaki Island. Ishigaki Island has a subtropical climate with high humidity (monthly average, 60.8-78.7%) and high temperature (monthly average, 18.5-29.4°C) throughout the year. METHODS: We diagnosed AD prevalence and analyzed eight FLG loss-of-function mutations in the Japanese population against a cohort of 721 children from the Kyushu University Ishigaki Atopic Dermatitis Study (KIDS) cohort. Parents gave consent for the mutation analysis during their medical examinations from 2001 to 2006. RESULTS: Average AD prevalence was 7.3% per year, and a total of 127 children (17.6%) were diagnosed with AD at least once between 2001 and 2006. The average total serum IgE level differed significantly between the AD and non-AD groups (199.0 and 69.0IU/ml, respectively). Although five kinds of FLG loss-of-function mutations isolated in previous Japanese FLG mutation studies were identified, the FLG loss-of-function mutation frequency in children of the KIDS cohort was not significantly different between the AD and non-AD groups (7.9% and 6.1%, respectively; P=0.174). CONCLUSION: The FLG loss-of-function mutation frequency was not significantly different between the AD and non-AD groups in a cohort of children from Ishigaki Island, which has a subtropical climate, suggesting that FLG loss-of-function mutations are not always a predisposing factor for AD prevalence.


Subject(s)
Dermatitis, Atopic/genetics , Genetic Predisposition to Disease , Intermediate Filament Proteins/genetics , Intermediate Filament Proteins/physiology , Mutation , Child , Child, Preschool , Cohort Studies , Dermatitis, Atopic/ethnology , Female , Filaggrin Proteins , Genotype , Humans , Immunoglobulin E/blood , Infant , Inflammation , Japan , Male , Odds Ratio , Prevalence , Skin/metabolism , Tropical Climate
10.
J Immunol Methods ; 412: 53-69, 2014 Oct.
Article in English | MEDLINE | ID: mdl-25010461

ABSTRACT

In vitro assembly of two or three PCR fragments using primers is a common method of constructing scFv fragments for display on the surface of phage. However, mismatch annealing often occurs during in this step, leading to cloning and display of incomplete Fab or scFv fragments. To overcome this limitation, we developed a ligation-based two-fragment assembly (LTFA) protocol that involved separately cloning VH and Vκ fragments into the high-copy-number plasmid pUC18. The VH and Vκ fragments had randomized complementarity-determining region 3 (CDR3) and were joined with a peptidyl linker composed of (G4S)3. Using this approach, complete sequences of scFv fragments were successfully constructed, and the sequencing of 83 scFv clones revealed that none of the sequences, including the linker region, contained deletions or mutations. In contrast, linker sequences generated using a conventional two-fragment PCR assembly (TFPA) protocol often contained sequence anomalies, including large truncations. Using the LTFA protocol, a final library size of 1.0×10(8)cfu was achieved. Examination of the amino acid profiles of the generated scFv fragments within the randomized regions introduced using degenerate codons did not detect any bias from that expected based on stochastic distribution. After several cycles of panning with this library, antigen-specific scFvs against two reference antigens, hen egg lysozyme and streptavidin were detected. In addition, scFvs with specificity against peptidyl antigens in the loop region of the Medaka ortholog of human C6orf89, which encodes a histone deacetylase enhancer that interacts with the bombesin receptor, were also obtained. The LTFA protocol developed here is robust and allows for the easy construction of integral scFv fragments compared with conventional TFPA. Utilizing LTFA, other CDRs can be readily combined. This approach also allows for the in vitro maturation of scFv fragments by separately introducing randomization in CDRs or using error-prone PCR for the amplification of pre-selected sequences as a template scaffold.


Subject(s)
DNA Shuffling/methods , Gene Library , Immunoglobulin Heavy Chains/genetics , Immunoglobulin Light Chains/genetics , Immunoglobulin Variable Region/genetics , Single-Chain Antibodies/genetics , Animals , Antibody Affinity , Cloning, Molecular , Humans , Mice , Muramidase/immunology , Oryzias , Proteins/genetics , Sequence Analysis, DNA , Streptavidin/immunology
11.
Transgenic Res ; 23(2): 317-29, 2014 Apr.
Article in English | MEDLINE | ID: mdl-24293126

ABSTRACT

Down syndrome (DS), also known as Trisomy 21, is the most common chromosome aneuploidy in live-born children and displays a complicated symptom. To date, several kinds of mouse models have been generated to understand the molecular pathology of DS, yet the gene dosage effects and gene(s)-phenotype(s) correlation are not well understood. In this study, we established a novel method to generate a partial trisomy mice using the mouse ES cells that harbor a single copy of human artificial chromosome (HAC), into which a small human DNA segment containing human chromosome 21 genes cloned in a bacterial artificial chromosome (BAC) was recombined. The produced mice were found to maintain the HAC carrying human genes as a mini-chromosome, hence termed as a Trans-Mini-Chromosomal (TMC) mouse, and HAC was transmitted for more than twenty generations independent from endogenous mouse chromosomes. The three human transgenes including cystathionine ß-synthase, U2 auxiliary factor and crystalline alpha A were expressed in several mouse tissues with various expression levels relative to mouse endogenous genes. The novel system is applicable to any of human and/or mouse BAC clones. Thus, the TMC mouse carrying a HAC with a limited number of genes would provide a novel tool for studying gene dosage effects involved in the DS molecular pathogenesis and the gene(s)-phenotype(s) correlation.


Subject(s)
Chromosomes, Artificial, Human/genetics , Chromosomes, Human, Pair 21/genetics , Disease Models, Animal , Down Syndrome/genetics , Animals , Crosses, Genetic , Embryonic Stem Cells/metabolism , Gene Dosage/genetics , Gene Expression Profiling , Humans , In Situ Hybridization, Fluorescence , Mice , Reverse Transcriptase Polymerase Chain Reaction , Transgenes/genetics
12.
Gene ; 530(1): 19-25, 2013 Nov 01.
Article in English | MEDLINE | ID: mdl-23954874

ABSTRACT

Although mutations of autoimmune regulator (AIRE) gene are responsible for autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), presenting a wide spectrum of many characteristic and non-characteristic clinical features, some patients lack AIRE gene mutations. Therefore, something other than a mutation, such as dysregulation of AIRE gene, may be a causal factor for APECED or its related diseases. However, regulatory mechanisms for AIRE gene expression and/or translation have still remained elusive. We found that IL-2-stimulated CD4(+) T (IL-2T) cells showed a high expression of AIRE gene, but very low AIRE protein production, while Epstein-Barr virus-transformed B (EBV-B) cells express both AIRE gene and AIRE protein. By using microarray analysis, we could identify miR-220b as a possible regulatory mechanism for AIRE gene translation in IL-2T cells. Here we report that miR-220b significantly reduced the expression of AIRE protein in AIRE gene with 3'UTR region transfected 293T cells, whereas no alteration of AIRE protein production was observed in the open reading frame of AIRE gene alone transfected cells. In addition, anti-miR-220b reversed the inhibitory function of miR-220b for the expression of AIRE protein in AIRE gene with 3'UTR region transfected cells. Moreover, when AIRE gene transfected cells with mutated 3'UTR were transfected with miR-220b, no reduction of AIRE protein production was observed. Taken together, it was concluded that miR-220b inhibited the AIRE gene translation through the 3'UTR region of AIRE gene, indicating that miR-220b could serve as a regulator for human AIRE gene translation.


Subject(s)
Interleukin-2/metabolism , MicroRNAs/genetics , Polyendocrinopathies, Autoimmune/genetics , Transcription Factors/genetics , CD4-Positive T-Lymphocytes/metabolism , Gene Expression Regulation/genetics , Herpesvirus 4, Human/genetics , Humans , Mutation , Polyendocrinopathies, Autoimmune/immunology , Polyendocrinopathies, Autoimmune/virology , Protein Biosynthesis , AIRE Protein
13.
Exp Cell Res ; 319(8): 1146-55, 2013 May 01.
Article in English | MEDLINE | ID: mdl-23499740

ABSTRACT

Recently, we isolated 4 distinct kinds of single chain antibody against human EGF receptor (EGFR) after screening the Keio phage display scFv library by using two methods of target-guided proximity labeling. In the current study, these monovalent scFv antibodies were converted to bivalent IgGs of humanized forms (hIgGs) by recombinant technology using the specially designed expression vectors followed by protein production in CHO cells. The resulting recombinant hIgGs were examined for their binding specificity using several different transformed human BJ cell lines that express deletion mutants of EGFR, each lacking one of 4 distinct extracellular domains (L1, L2, C1 and C2). Immuno-fluorescent microscopy and immuno-precipitation assay on these cells indicated that 4 distinct kinds of hIgGs bind to one of 3 different domains (L1, C1 and C2). Then, these hIgGs were further examined for biological effects on human A431 cancer cells, which overexpress EGFR. The results indicated that hIgG38 binding to L1 and hIgG45 binding to C2 substantially suppressed the EGF-induced phosphorylation of EGFR, resulting in the growth inhibition of A431 cancer cells. On the contrary, hIgG40 binding to C1 and hIgG42 binding to another site (epitope) of C2 exhibited no such inhibitory effects. Thus, the newly produced four recombinant hIgG antibodies recognize 4 different sites (epitopes) in 3 different extracellular domains of EGFR and exhibit different biological effects on cancer cells. These characteristics are somewhat different from the currently utilized therapeutic anti-EGFR antibodies. Hence, these hIgG antibodies will be invaluable as a research tool for the detailed molecular analysis of the EGFR-mediated signal transduction mechanism and more importantly a possible application as new therapeutic agents to treat certain types of cancers.


Subject(s)
Cell Proliferation/drug effects , ErbB Receptors/immunology , ErbB Receptors/metabolism , Immunoglobulin G/metabolism , Immunoglobulin G/pharmacology , Neoplasms/pathology , Animals , Antibodies, Monoclonal/metabolism , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal/therapeutic use , Antibody Specificity , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , CHO Cells , Cell Line, Tumor , Cricetinae , Cricetulus , Epitopes/immunology , Epitopes/metabolism , ErbB Receptors/chemistry , Extracellular Space , Humans , Immunoglobulin G/therapeutic use , Immunotherapy/methods , Neoplasms/therapy , Protein Binding , Protein Structure, Tertiary , Recombinant Proteins/metabolism , Recombinant Proteins/pharmacology , Recombinant Proteins/therapeutic use
14.
Transgenic Res ; 22(2): 435-44, 2013 Apr.
Article in English | MEDLINE | ID: mdl-22983842

ABSTRACT

The precise control of spatiotemporal expression of target genes is crucial when establishing transgenic animals, and the introduction of genes for fluorescent marker proteins is inevitable for accelerating research at molecular levels. To assist this, we constructed a novel dual promoter expression vector for two independent reporter genes, green fluorescent protein (GFP) and red fluorescent protein (mCherry). Their expression is designed under the control of two distinct tissue-specific promoters, e.g. zebrafish cardiac muscle-specific promoter (cmlc2) and medaka skeletal muscle-specific promoter (myl2) derived from the myosin light chain 2 genes, and they are placed in a head-to-head orientation. After microinjecting the dual promoter expression vector into fertilized eggs of medaka, the developing fish embryos and the resulting transgenic fish lines showed strong GFP signal in the whole body (skeletal muscle) and mCherry signal in the heart (cardiac muscle). However, weak GFP signal was observed in the heart, indicating a leakiness of the skeletal muscle promoter. To improve the stringency of dual promoter expression, we inserted two chicken-derived insulators, e.g. tandem copies of the core sequence (250 bp) of cHS4 (5'-hypersensitive site-4 chicken beta-globin insulator), in the boundary of two promoters. The dual promoter expression vector with insulator now ensured the stringent tissue-specific expression in the transgenic fish lines. Thus, our dual promoter expression system with insulator is compatible to the conventional IRES and fused reporter gene systems and will be an alternative method to produce the transgenic fishes.


Subject(s)
Animals, Genetically Modified/growth & development , Green Fluorescent Proteins/genetics , Luminescent Proteins/genetics , Organ Specificity/genetics , Promoter Regions, Genetic , Animals , Chickens/genetics , Gene Expression Regulation, Developmental , Insulator Elements/genetics , Oryzias/genetics , Zebrafish/genetics , Zebrafish/growth & development , Red Fluorescent Protein
15.
Gene ; 511(2): 202-17, 2012 Dec 15.
Article in English | MEDLINE | ID: mdl-23036704

ABSTRACT

Through the comprehensive analysis of the genomic DNA sequence of human chromosome 22, we identified a novel gene of 702 kb encoding a big protein of 2481 amino acid residues, and named it as TPRBK (TPR containing big gene cloned at Keio). A novel protein TPRBK possesses 25 units of the TPR motif, which has been known to associate with a diverse range of biological functions. Orthologous genes of human TPRBK were found widely in animal species, from insecta to mammal, but not found in plants, fungi and nematoda. Northern blotting and RT-PCR analyses revealed that TPRBK gene is expressed ubiquitously in the human and mouse fetal tissues and various cell lines of human, monkey and mouse. Immunofluorescent staining of the synchronized monkey COS-7 cells with several relevant antibodies indicated that TPRBK changes its subcellular localization during the cell cycle: at interphase TPRBK locates on the centrosomes, during mitosis it translocates from spindle poles to mitotic spindles then to spindle midzone, and through a period of cytokinesis it stays on the midbody. Co-immunoprecipitation assay and immunofluorescent staining with adequate antibodies revealed that TPRBK binds to Aurora B, and those proteins together translocate throughout mitosis and cytokinesis. Treatments of cells with two drugs (Blebbistatin and Y-27632), that are known to inhibit the contractility of actin-myosin, disturbed the proper intracellular localization of TPRBK. Moreover, the knockdown of TPRBK expression by small interfering RNA (siRNA) suppressed the bundling of spindle midzone microtubules and disrupted the midbody formation, arresting the cells at G(2)+M phase. These observations indicated that a novel big protein TPRBK is essential for the formation and integrity of the midbody, hence we postulated that TPRBK plays a critical role in the progress of mitosis and cytokinesis during mammalian cell cycle.


Subject(s)
Cell Cycle Proteins/physiology , Cytokinesis/physiology , Mitosis/physiology , Animals , Blotting, Northern , Blotting, Western , COS Cells , Cell Cycle Proteins/genetics , Chlorocebus aethiops , DNA, Complementary , Flow Cytometry , Fluorescent Antibody Technique, Indirect , Humans , Polymerase Chain Reaction , RNA, Small Interfering , Subcellular Fractions/metabolism
16.
Genomics ; 100(2): 81-92, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22659240

ABSTRACT

We have developed a graphical image database CancerProView (URL: http://cancerproview.dmb.med.keio.ac.jp/php/cpv.html) to assist the search for alterations of the motifs/domains in the cancer-related proteins that are caused by mutations in the corresponding genes. For the CancerProView, we have collected various kinds of data on 180 cancer-related proteins in terms of the motifs/domains, genomic structures of corresponding genes, and 109 charts of the protein interaction pathways. Moreover, we have collected the relevant data on 1041 reference genes including 197 non-cancer disease-associated genes, and the nucleotide sequences for 2011 full-length cDNA's and the alternatively spliced transcript variants. Thus, the CancerProView database system would provide valuable information to facilitate basic cancer research as well as for designing new molecular diagnosis and drug discovery for cancers. The CancerProView database can be operated via Internet with any Web browser, and the system is freely available to interested users without ID and password.


Subject(s)
Databases, Genetic , Genes, Neoplasm , Neoplasm Proteins/genetics , Neoplasms/genetics , Genetic Predisposition to Disease , Humans , Internet , Mutation , Neoplasms/diagnosis , User-Computer Interface
17.
J Surg Res ; 176(1): 293-300, 2012 Jul.
Article in English | MEDLINE | ID: mdl-21962809

ABSTRACT

BACKGROUND: Interleukin-18 (IL-18) is a potent proinflammatory cytokine that augments both innate and acquired immune responses. It is also a crucial regulator of lymphocyte production of interferon-γ (IFN-γ), which can promote acute cellular rejection of transplanted solid organs. METHODS: To evaluate the role of IL-18 in liver transplantation, we constructed an adenoviral vector encoding IL-18 binding protein (Adex-IL18bp), which specifically suppressed the biologic activity of IL-18, and examined the effect of this suppression on liver allografts by using a high-responder rat model (ACI to Lewis) of orthotopic liver transplantation (OLTx). Donor rats were given one intravenous injection of Adex-IL18bp or Adex-LacZ (control vector) 2 d before OLTx. RESULTS: Seven days after OLTx, overexpression of IL-18bp resulting from the adenovirus gene transfer was associated with significantly decreased serum alanine aminotransferase levels and less histologic hepatic injury in recipient rats with Adex-IL18bp-pretreated donors compared with Adex-LacZ controls. Adex-IL18bp pretreatment also significantly prolonged rat/allograft survival, inhibited expression of IFN-γ, and reduced levels (versus control values) of both CXCL10 and CX3CL1, which can be induced by IFN-γ. CONCLUSION: These results suggest that IL-18 has an important role in liver allograft rejection through IFN-γ and chemokines and that specific suppression of IL-18 may improve liver function early after transplantation.


Subject(s)
Graft Survival/physiology , Interleukin-18/physiology , Liver Transplantation/physiology , Liver/metabolism , Adenoviridae/genetics , Alanine Transaminase/blood , Animals , Chemokines/metabolism , Graft Survival/drug effects , Intercellular Signaling Peptides and Proteins/genetics , Intercellular Signaling Peptides and Proteins/pharmacology , Interferon-gamma/metabolism , Interleukin-18/antagonists & inhibitors , Liver/drug effects , Male , Models, Animal , Rats , Rats, Inbred Lew , Transplantation, Homologous
18.
J Immunol Methods ; 372(1-2): 127-36, 2011 Sep 30.
Article in English | MEDLINE | ID: mdl-21782821

ABSTRACT

We recently constructed the scFv-displaying phage library with extremely high repertoire and have successfully utilized for screening scFv antibodies against various proteins, polysaccharides and glyco-lipids. Here, we developed a new screening strategy to isolate scFv antibodies against cell surface EGF receptor (EGFR). For this, we applied two slightly different methods of "target-guided proximity labeling," such as Proximity selection (ProxiMol) method and a new sulfo-SBED labeling method with the aide of monoclonal anti-human EGFR antibody B4G7 as a guide molecule. ProxiMol method relies on the Biotin-labeling of scFv-displaying phages that bound to the target in a vicinity of 100Å from the guide molecule, whereas sulfo-SBED method transfers Biotin to scFv-displaying phages, which bound to the target in a distance of 20 Å. After two rounds of panning on the EGFR-overexpressing A431 cells starting from approx. 1 × 10¹² pfu, 47 each of Biotin-labeled scFv-displaying phages were recovered using Streptoavidin-coated magnetic beads, and among them total 11 scFv-phages were found to be definitely positive for binding to A431 cell surface by ELISA assay. Restriction mapping and sequencing analysis of these scFv-phage DNAs revealed that they encode 4 different scFv-nucleotide sequences in total. Immuno-fluorescent microscopy provided evidence that these 4 scFv antibodies bind specifically to EGFR on the A431 cells, showing slightly different staining patterns. Thus, "target-guided proximity labeling" methods were powerful for isolating scFv-displaying phages that recognize distinct extracellular domains of the target receptor. This novel screening strategy could be applicable to many other cell surface antigens and receptors.


Subject(s)
ErbB Receptors/immunology , Immunoglobulin Fragments/immunology , Single-Chain Antibodies/immunology , Antibody Specificity , Cell Line, Tumor , Cloning, Molecular , Epitopes/immunology , Humans , Immunoglobulin Fragments/genetics , Peptide Library
19.
Exp Cell Res ; 317(14): 2019-30, 2011 Aug 15.
Article in English | MEDLINE | ID: mdl-21683072

ABSTRACT

In the human thymus, AIRE (autoimmune regulator) gene is expressed in a very limited type of medullary thymic epithelial cells (mTECs) and no cognate cell lines are available, hence the molecular analysis of AIRE gene function has been difficult. To improve this situation, we attempted to isolate Aire-expressing cells and established three cell lines (Aire⁺TEC1, Aire⁺TEC2, Aire⁺DC) from the abnormally enlarged thymus, which was developed in the transgenic mice expressing SV40 T-antigen driven by the mouse Aire gene promoter. When these Aire⁺ cell lines were co-cultured with fresh thymocytes, they adhered to the majority of thymocytes and induced apoptosis as if negative selection of T-cells in the thymus is occurring in vitro. Further analysis revealed that these Aire⁺ cell lines are derived from mTECs and exhibit characteristic natures of "antigen presenting cells" including several distinct abilities: to express a variety of peripheral tissue-specific antigens, to produce immunoproteasome and immunological synapse, and to express some of TNFSFs (tumor necrosis factor super families). Thus, the newly established Aire⁺ cell lines will be invaluable for the further detailed analysis of AIRE gene function in the central tolerance of immunity and autoimmune disease.


Subject(s)
Antigens/biosynthesis , Epithelial Cells/immunology , Epithelial Cells/metabolism , T-Lymphocytes/cytology , T-Lymphocytes/immunology , Thymus Gland/cytology , Animals , Antigens/genetics , Antigens/immunology , Cells, Cultured , Epithelial Cells/cytology , Mice , Mice, Inbred C57BL , Mice, Transgenic , Organ Specificity , Transcription Factors/genetics , Transcription Factors/immunology , AIRE Protein
20.
J Invest Dermatol ; 131(2): 410-7, 2011 Feb.
Article in English | MEDLINE | ID: mdl-21048786

ABSTRACT

In the mechanism of thymus-induced central tolerance, the transcription factor Aire has been demonstrated to promote the expression of a wide range of peripheral organ-specific antigens (Ags) in the medullary thymic epithelial cells (mTECs), which serve as self-Ags in negative selection. We examined the expression of desmoglein 3 (Dsg3), the autoantigen in pemphigus vulgaris (PV), in mouse thymus and the involvement of Aire in tolerance to Dsg3. Immunofluorescence and in situ hybridization revealed Dsg3 in single cells or in clusters in ∼3% of mTECs near the cortico-medullary junction of the thymus in C57BL/6 mice. Dsg3-expressing mTECs also expressed some Ags of skin-unrelated peripheral organs simultaneously. In contrast, Dsg3-positive mTECs were not detected in the Aire(-/-) thymus. Adoptive transfer of splenocytes from Aire(-/-) mice immunized with Dsg3 did not induce anti-Dsg3 IgG production or PV phenotype in Rag2(-/-) recipient mice. However, Aire(-/-) CD4(+) T cells, but not Aire(+/+) CD4(+) T cells, induced low levels of anti-Dsg3 IgG production when transferred with Dsg3(-/-) B cells. These findings indicate that Aire has an important role in Dsg3 expression as well as in selection of T cells that help B cells to produce anti-Dsg3 IgG in thymus.


Subject(s)
Autoantigens/metabolism , CD4-Positive T-Lymphocytes/immunology , Desmoglein 3/metabolism , Immune Tolerance/physiology , Pemphigus/metabolism , Thymus Gland/metabolism , Transcription Factors/metabolism , Adaptive Immunity/physiology , Animals , Antibodies/immunology , Antibodies/metabolism , Autoantigens/genetics , Autoantigens/immunology , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , CD4-Positive T-Lymphocytes/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Desmoglein 3/genetics , Desmoglein 3/immunology , Epithelial Cells/immunology , Epithelial Cells/metabolism , Epithelial Cells/pathology , Immunoglobulin G/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Models, Animal , Pemphigus/immunology , Thymus Gland/immunology , Thymus Gland/pathology , Transcription Factors/genetics , AIRE Protein
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