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1.
Sci Rep ; 11(1): 19661, 2021 10 04.
Article in English | MEDLINE | ID: mdl-34608183

ABSTRACT

Genetic information is protected against a variety of genotoxins including ionizing radiation (IR) through the DNA double-strand break (DSB) repair machinery. Genome-wide association studies and clinical sequencing of cancer patients have suggested that a number of variants in the DNA DSB repair genes might underlie individual differences in chromosomal radiosensitivity within human populations. However, the number of established variants that directly affect radiosensitivity is still limited. In this study, we performed whole-exome sequencing of 29 Japanese ovarian cancer patients and detected the NBS1 I171V variant, which is estimated to exist at a rate of approximately 0.15% in healthy human populations, in one patient. To clarify whether this variant indeed contributes to chromosomal radiosensitivity, we generated NBS1 I171V variant homozygous knock-in HCT116 cells and mice using the CRISPR/Cas9 system. Radiation-induced micronucleus formation and chromosomal aberration frequency were significantly increased in both HCT116 cells and mouse embryonic fibroblasts (MEFs) with knock-in of the NBS1 I171V variant compared with the levels in wild-type cells. These results suggested that the NBS1 I171V variant might be a genetic factor underlying individual differences in chromosomal radiosensitivity.


Subject(s)
Alleles , Amino Acid Substitution , Biological Variation, Population/genetics , Cell Cycle Proteins/genetics , Chromosomal Instability/radiation effects , Mutation , Nuclear Proteins/genetics , Radiation Tolerance/genetics , Binding Sites , Biomarkers, Tumor , Cell Cycle Proteins/metabolism , Cell Line, Tumor , DNA Copy Number Variations , Female , Gene Editing , Gene Knock-In Techniques , Genetic Predisposition to Disease , Humans , Nuclear Proteins/metabolism , Ovarian Neoplasms/genetics , Ovarian Neoplasms/radiotherapy , Protein Binding , Radiation, Ionizing
2.
Cells ; 9(1)2020 01 17.
Article in English | MEDLINE | ID: mdl-31963583

ABSTRACT

Chromosomal segregation errors in germ cells and early embryonic development underlie aneuploidies, which are numerical chromosomal abnormalities causing fetal absorption, developmental anomalies, and carcinogenesis. It has been considered that human aneuploidy disorders cannot be resolved by radical treatment. However, recent studies have demonstrated that aneuploidies can be rescued to a normal diploid state using genetic engineering in cultured cells. Here, we summarize a series of studies mainly applying genome editing to eliminate an extra copy of human chromosome 21, the cause of the most common constitutional aneuploidy disorder Down syndrome. We also present findings on induced pluripotent stem cell reprogramming, which has been shown to be one of the most promising technologies for converting aneuploidies into normal diploidy without the risk of genetic alterations such as genome editing-mediated off-target effects.


Subject(s)
Cellular Reprogramming Techniques/methods , Chromosome Disorders/genetics , Chromosome Disorders/therapy , Down Syndrome/genetics , Gene Editing/methods , Induced Pluripotent Stem Cells/metabolism , Sex Chromosomes/genetics , Trisomy/genetics , Aneuploidy , CRISPR-Cas Systems , Chromosome Disorders/metabolism , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Down Syndrome/therapy , Humans , Induced Pluripotent Stem Cells/cytology , Mosaicism , Sex Chromosomes/pathology
3.
J Arrhythm ; 33(6): 643-645, 2017 Dec.
Article in English | MEDLINE | ID: mdl-29255517

ABSTRACT

A 53-year-old woman reported burning pain, muscle weakness, and dysesthesia of the left arm 2 months after permanent pacemaker insertion in the ipsilateral side for the treatment of sick sinus syndrome. Complex regional pain syndrome (CRPS) induced by pacemaker implantation was diagnosed. In 2017, her pulse generator became exhausted and was exchanged carefully to avoid exacerbation of CRPS, under the application of local anesthesia and premedication. Six months later, the patient's grip strength in her left hand remained lower relative to that in her right hand. Although rare, the presence of CRPS following device implantation should be remembered.

4.
J Arrhythm ; 33(5): 424-429, 2017 Oct.
Article in English | MEDLINE | ID: mdl-29021844

ABSTRACT

Acetylcholine is widely used for a diagnostic provocation test of coronary spasm in patients with vasospastic angina. Acetylcholine usually induces coronary vasodilatation mediated by muscarinic receptor activation, but sometimes it evokes vasoconstriction of coronary arteries where the endothelium is damaged. Early repolarization syndrome is characterized by a J wave observed at the end of the QRS complex in a surface electrocardiogram. The J wave is attributed to the transmural voltage gradient at the early repolarization phase across the ventricular wall, which stems mainly from prominent transient outward current in the epicardium, but not in the endocardium. Transient high-dose application of acetylcholine into the epicardial coronary arteries provides a unique opportunity to augment net outward current, selectively, in the ventricular epicardium and unmask the J wave, irrespective of the cardiac ischemia based on coronary spasm. Acetylcholine augments cardiac membrane potassium conductance by enhancing acetylcholine-activated potassium current directly and by activating adenosine triphosphate-sensitive potassium current, in addition to the reduced sodium and calcium currents in the setting of severe ischemia due to vasospasm. However, the role of acetylcholine as an arrhythmogenic probe of the J wave induction in patients with suspected early repolarization syndrome warrants future prospective study.

5.
J Arrhythm ; 33(3): 234-236, 2017 Jun.
Article in English | MEDLINE | ID: mdl-28607620

ABSTRACT

A 51-year-old man with a resuscitation episode was referred to our hospital. Coronary angiography revealed a focal spasm overlapped with organic stenosis where a bare metal stent was implanted. Acetylcholine (ACh) provocation test did not induce chest pain. It revealed no discernible ST-T changes but unmasked a J wave at the end of the QRS complex, which was associated with short-coupled repetitive premature ventricular beats. A J wave reportedly appears immediately before the onset of ventricular fibrillation caused by vasospastic angina. However, a J wave observed newly after a coronary spasm provocation test using ACh without ST-T changes is informative when considering the mechanisms of the J wave.

7.
Proc Natl Acad Sci U S A ; 109(27): 10915-20, 2012 Jul 03.
Article in English | MEDLINE | ID: mdl-22711830

ABSTRACT

To understand complex biological systems, such as the development of multicellular organisms, it is important to characterize the gene expression dynamics. However, there is currently no universal technique for targeted insertion of reporter genes and quantitative imaging in multicellular model systems. Recently, genome editing using zinc-finger nucleases (ZFNs) has been reported in several models. ZFNs consist of a zinc-finger DNA-binding array with the nuclease domain of the restriction enzyme FokI and facilitate targeted transgene insertion. In this study, we successfully inserted a GFP reporter cassette into the HpEts1 gene locus of the sea urchin, Hemicentrotus pulcherrimus. We achieved this insertion by injecting eggs with a pair of ZFNs for HpEts1 with a targeting donor construct that contained ∼1-kb homology arms and a 2A-histone H2B-GFP cassette. We increased the efficiency of the ZFN-mediated targeted transgene insertion by in situ linearization of the targeting donor construct and cointroduction of an mRNA for a dominant-negative form of HpLig4, which encodes the H. pulcherrimus homolog of DNA ligase IV required for error-prone nonhomologous end joining. We measured the fluorescence intensity of GFP at the single-cell level in living embryos during development and found that there was variation in HpEts1 expression among the primary mesenchyme cells. These findings demonstrate the feasibility of ZFN-mediated targeted transgene insertion to enable quantification of the expression levels of endogenous genes during development in living sea urchin embryos.


Subject(s)
Embryo, Nonmammalian/physiology , Gene Expression Regulation, Developmental/physiology , Genes, Reporter/genetics , Sea Urchins/genetics , Zinc Fingers/genetics , Amino Acid Sequence , Animals , DNA Ligase ATP , DNA Ligases/genetics , Deoxyribonucleases, Type II Site-Specific/genetics , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/embryology , Green Fluorescent Proteins/genetics , Mesoderm/cytology , Mesoderm/physiology , Molecular Biology/methods , Molecular Sequence Data , Phenotype , Primary Cell Culture , Sea Urchins/embryology , Transgenes/genetics
8.
J Med Virol ; 84(3): 479-86, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22246835

ABSTRACT

A novel detection system was established previously for cells infected with the human cytomegalovirus (HCMV) in vitro that utilizes the unique IE1-dependent nuclear dispersion of promyelocytic leukemia (PML) bodies early in the HCMV replication cycle. This assay system, designated "the PML assay," makes use of the GFP-PML-expressing cell line SE/15, and allows real-time monitoring of infected cells by fluorescence microscopy without any staining procedures. A rapid and quantitative drug susceptibility testing was developed for low-titer clinical isolates propagated in fibroblasts in vitro. The present study sought to exploit the PML assay for evaluating in vivo status of HCMV without virus isolation. Progeny viruses were detected directly from peripheral blood mononuclear cells (PBMCs) infected in vivo obtained from hematopoietic stem cell transplantation recipients. The overall positivity of the PML assay tended to correlate with the levels of genomic DNA. Direct phenotypic susceptibility testing detected one ganciclovir (GCV)-resistant case among 19 samples, which was confirmed further by genomic and plaque reduction assays. However, in another patient with the sequence-proven mutant confirmed by sequencing, the progeny viruses exhibiting GCV-resistance were not detected. Studies on the isolated virus from the latter patient suggested the possibility that replication efficiency may differ between PBMCs and lesions infected in vivo, which may hamper the detection of GCV-resistant viruses by the PML assay, at least in this case. Taken together, the PML assay is sufficiently sensitive to monitor replication-competent HCMV directly from PBMCs infected in vivo, and provides a novel tool for comparing the characteristics of HCMV strains infected in vivo.


Subject(s)
Cytomegalovirus/isolation & purification , Hematopoietic Stem Cell Transplantation , Leukocytes, Mononuclear/virology , Adolescent , Adult , Aged , Cell Line , Cytomegalovirus/drug effects , Female , Ganciclovir/pharmacology , Humans , Leukocytes, Mononuclear/drug effects , Male , Microbial Sensitivity Tests , Middle Aged , Virology/methods , Virus Replication/drug effects , Young Adult
9.
Dev Genes Evol ; 221(3): 157-66, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21706447

ABSTRACT

Sulfatases such as arylsulfatase and heparan sulfate 6-O-endosulfatase play important roles in morphogenesis during sea urchin development. For the activation of these sulfatases, Cα-formylglycine formation by sulfatase modifying factor (Sumf) is required. In this study, to clarify the regulatory mechanisms for the activation of sulfatases during sea urchin development, we examined the expression and function of the Hemicentrotus pulcherrimus homologs of Sumf1 and Sumf2 (HpSumf1 and HpSumf2, respectively). Expression of HpSumf1 but not HpSumf2 mRNA was dynamically changed during early development. Functional analyses of recombinant HpSumf1 and HpSumf2 using HEK293T cells expressing mouse arylsulfatase A (ArsA) indicated that HpSumf1 and HpSumf2 were both able to activate mammalian ArsA. Knockdown of HpSumf1 using morpholino antisense oligonucleotides caused abnormal spicule formation in the sea urchin embryo. Injection of HpSumf2 mRNA had no effect on skeletogenesis, while injection of HpSumf1 mRNA induced severe supernumerary spicule formation. Taken together, these findings suggest that HpSumf1 is involved in the activation of sulfatases required for control of skeletogenesis.


Subject(s)
Hemicentrotus/embryology , Hemicentrotus/enzymology , Skeleton , Sulfatases/genetics , Amino Acid Sequence , Animals , Cells, Cultured , Cerebroside-Sulfatase/genetics , Cerebroside-Sulfatase/metabolism , Cloning, Molecular , Embryo, Nonmammalian , Enzyme Activation , Gene Expression Regulation, Developmental , Hemicentrotus/genetics , Mice , Molecular Sequence Data , Oligonucleotides, Antisense , Oxidoreductases Acting on Sulfur Group Donors , RNA, Messenger , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Homology, Amino Acid , Sulfatases/chemistry , Sulfatases/metabolism
10.
Genes Cells ; 15(8): 875-85, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20604805

ABSTRACT

We showed that engineered zinc-finger nucleases (ZFNs), which consist of a zinc-finger DNA-binding array and a nuclease domain of the restriction enzyme FokI, can introduce mutations at a specific genomic site in the sea urchin embryo. Using bacterial one-hybrid screening with zinc-finger randomized libraries and a single-strand annealing assay in cultured cells, ZFNs targeting the sea urchin Hemicentrotus pulcherrimus homologue of HesC (HpHesC) were efficiently selected. Consistent with the phenotype observed in embryos injected with an antisense morpholino oligonucleotide against HpHesC, an increase in the primary mesenchyme cell population was observed in embryos injected with a pair of HpHesC ZFN mRNAs. In addition, sequence analysis of the mutations showed that deletions and insertions occurred at the HpHesC target site in the embryos injected with the HpHesC ZFN mRNAs. These results suggest that targeted gene disruption using ZFNs is feasible for the sea urchin embryo.


Subject(s)
Endonucleases/chemistry , Endonucleases/metabolism , Hemicentrotus/embryology , Hemicentrotus/genetics , Mutagenesis, Site-Directed/methods , Zinc Fingers , Animals , Cells, Cultured , Endonucleases/genetics , Gene Library , Genetic Engineering , RNA, Messenger/genetics
11.
Connect Tissue Res ; 51(5): 388-96, 2010 Oct.
Article in English | MEDLINE | ID: mdl-20297902

ABSTRACT

Inherited deficiency for arylsulfatase (Ars) leads to lysosomal storage of sulfated compounds and to serious diseases such as growth retardation, heart failure, and demyelination in the central nervous system. Ars has been regarded as a lysosomal enzyme because of its hydrolytic activity on synthetic aromatic substrates and the lysosomal localization of its enzymatic activity. We previously demonstrated that a large portion of the mammalian arylsulfatase A (ArsA) protein exists on the cell surface of vascular endothelial cells, suggesting that ArsA plays a role in the components of the extracellular matrix. Here we show that ArsA functions as a substrate on which cells adhere and form protrusions. Coating culture plates with recombinant mouse ArsA (rmArsA) stimulates adhesion of human microvascular endothelial cells to the plate followed by the formation of cell protrusions as well as lamellipodia. rmArsA affects the architecture of the cytoskeleton, with a high density of actin filaments localized to peripheral regions of the cells and the extension of bundles of microtubules into the tips of cellular protrusions. rmArsA also affects the distribution pattern of the cell adhesion-associated proteins, integrin α2ß1, and paxillin. rmArsA seems to modulate signaling of basic fibroblast growth factor (bFGF) stimulating cytoskeletal rearrangement. We also show that rmArsA tightly binds to sulfated polysaccharides. We suggest that mammalian ArsA plays a role as a novel component of the extracellular matrix. This viewpoint of Ars could be very useful for clarifying the mechanisms underpinning syndromes caused by the deficiency of the function of Ars genes.


Subject(s)
Cerebroside-Sulfatase/physiology , Endothelial Cells/enzymology , Endothelial Cells/pathology , Extracellular Matrix Proteins/physiology , Extracellular Matrix/enzymology , Animals , Cell Line , Cerebroside-Sulfatase/deficiency , Cerebroside-Sulfatase/genetics , Extracellular Matrix/genetics , Extracellular Matrix/pathology , Extracellular Matrix Proteins/deficiency , Extracellular Matrix Proteins/genetics , Humans , Infant, Newborn , Male , Mice
12.
Mech Dev ; 127(3-4): 235-45, 2010 Apr.
Article in English | MEDLINE | ID: mdl-20036737

ABSTRACT

Cell surface heparan sulfate proteoglycans (HSPGs) play significant roles in the regulation of developmental signaling, including vascular endothelial growth factor (VEGF), fibroblast growth factor, Wnt and bone morphogenetic protein signaling, through modification of their sulfation patterns. Recent studies have revealed that one of the functions of heparan sulfate 6-O-endosulfatase (Sulf) is to remove the sulfate from the 6-O position of HSPGs at the cell surface, thereby regulating the binding activities of heparan sulfate (HS) chains to numerous ligands and receptors in animal species. In this study, we focused on the sea urchin Hemicentrotus pulcherrimus homolog of Sulf (HpSulf), and analyzed its expression pattern and functions during development. HpSulf protein was present throughout development and localized at cell surface of all blastomeres. In addition, the HS-specific epitope 10E4 was detected at the cell surface and partially colocalized with HpSulf. Knockdown of HpSulf using morpholino antisense oligonucleotides (MO) caused abnormal morphogenesis, and the development of MO-injected embryos was arrested before the hatched blastula stage, indicating that HpSulf is necessary for the early developmental process of sea urchin embryos. Furthermore, we found that injection of HpSulf mRNA suppressed the abnormal skeleton induced by overexpression of HpVEGF mRNA, whereas injection of an inactive form of HpSulf mRNA, containing mutated cysteines in the sulfatase domain, did not have this effect. Taken together, these results suggest that HpSulf is involved in the regulation of various signal transductions, including VEGF signaling, during sea urchin development.


Subject(s)
Sea Urchins/embryology , Signal Transduction , Sulfotransferases/metabolism , Vascular Endothelial Growth Factor A/metabolism , Amino Acid Sequence , Animals , Gene Knockdown Techniques , Molecular Sequence Data , Protein Conformation , RNA, Messenger/genetics , Sequence Homology, Amino Acid , Sulfotransferases/chemistry , Sulfotransferases/genetics
13.
Zoolog Sci ; 26(10): 686-90, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19832680

ABSTRACT

In sea urchin embryos, Notch signaling is required to segregate non-skeletogenic mesoderm from early endomesoderm, and is involved in endoderm development. To further investigate the role of Notch signaling in the endoderm cell lineage, we cloned a cDNA for the Hemicentrotus pulcherrimus ortholog of Suppressor of Hairless (HpSu(H)), which is a major mediator of the Notch signaling pathway, examined the expression during development and performed a functional analysis. HpSu(H) mRNA was ubiquitously expressed up to the unhatched blastula stage, and expression was exclusively detected in the vegetal plate region from the hatched blastula stage and then in the archenteron at the gastrula stage. Perturbation of HpSu(H) by injection of the dominant negative form of HpSu(H) (dn-HpSu(H)) mRNA into fertilized eggs led to the disappearance of secondary mesenchyme cells at the tip of the archenteron in the gastrula and pigment cells in the pluteus larva, confirming that Notch signaling is required for non-skeletogenic me soderm specification. In addition, injection of relatively high amounts of dn-HpSu(H) mRNA caused a defect or atrophy of the foregut in the archenteron at the pluteus stage. This result strongly suggests that Notch signaling is involved in foregut development during sea urchin development.


Subject(s)
Gastrointestinal Tract/embryology , Gene Expression Regulation, Developmental/physiology , Sea Urchins/embryology , Trans-Activators/metabolism , Aggression , Amino Acid Sequence , Animals , Molecular Sequence Data , Trans-Activators/genetics
14.
Dev Dyn ; 238(10): 2511-21, 2009 Oct.
Article in English | MEDLINE | ID: mdl-19705446

ABSTRACT

The nanos genes play important roles in the development of primordial germ cells in animal species. In the sea urchin, Hemicentrotus pulcherrimus, small micromere descendants specifically express HpNanos mRNA and this expression continues in the left coelomic pouch, which produces the major component of the adult rudiment. In this study, we showed that morpholino knockdown of HpNanos resulted in a delay of primary mesenchyme cell ingression and a decrease in the number of cells comprising the left coelomic pouch. Knockdown analysis in chimeras and whole embryos revealed the disappearance of small micromere descendants from the archenteron tip. Furthermore, the expression of HpNanos mRNA was induced in other cell lineages in the HpNanos-knockdown and micromere-deleted embryos. Taken together, our results suggest that HpNanos is involved in the inductive interaction of small micromere descendants with other cell lineages, and that HpNanos is required for the survival of small micromere descendants.


Subject(s)
RNA-Binding Proteins/metabolism , Sea Urchins/embryology , Sea Urchins/metabolism , Amino Acid Chloromethyl Ketones/metabolism , Animals , Apoptosis/physiology , Cell Lineage , Cell Proliferation , Chimera , Cysteine Proteinase Inhibitors/metabolism , Embryo, Nonmammalian/cytology , Embryo, Nonmammalian/physiology , Gene Expression Regulation, Developmental , Morphogenesis/physiology , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Sea Urchins/anatomy & histology
15.
Spine (Phila Pa 1976) ; 28(13): 1379-84, 2003 Jul 01.
Article in English | MEDLINE | ID: mdl-12838094

ABSTRACT

STUDY DESIGN: An anatomic study investigated the cervical dorsal rami and major cervical paravertebral muscles. OBJECTIVE: To provide a detailed description of the cervical dorsal rami and important paravertebral muscles as a way of avoiding inadvertent injuries during the posterior approach. SUMMARY OF BACKGROUND DATA: No detailed anatomic studies of the nerves and the muscles in the posterior neck useful for the posterior approach have been reported previously. METHODS: Running courses of the cervical dorsal rami of spinal nerves and the morphology of cervical major paravertebral muscles were studied using 14 cadavers. In four posterior approaches of cervical laminoplasty, subcutaneous facial exits of cutaneous nerves and the running course of the right C3 medial branches around facet joint were exposed for observation of living anatomy. RESULTS: Every medial branch from the dorsal rami of the C3-C8 spinal nerves passed through an anatomic tunnel dorsolateral to the facet joint. The base of the tunnel was a bony gutter between neighboring facet joint capsules, and the roof was the tendon of the semispinalis capitis. In this tunnel, the medial branch had a little laxity in moving, and was assumed to be the most susceptible to iatrogenic injury during the operation. The semispinalis cervicis was composed with long muscle bundles. Each of these had only one or two innervating nerves from the dorsal rami of cervical spinal nerves. Cutaneous branches from the dorsal rami were found adjacent to every spinous process below the C2 spinous process in cadaveric studies. However, only two or three larger cutaneous nerves were discernible below the C5 or C6 spinous process in surgical approaches. CONCLUSIONS: With the posterior approach to the cervical spine, a precise knowledge of the cervical dorsal rami anatomy and the innervating patterns of the paravertebral muscles is necessary for avoidance of inadvertent injuries to the nerves.


Subject(s)
Cervical Vertebrae/anatomy & histology , Muscle, Skeletal/anatomy & histology , Neck/anatomy & histology , Nerve Fibers , Spine/anatomy & histology , Adolescent , Adult , Aged , Cadaver , Cervical Vertebrae/innervation , Female , Humans , Male , Medical Errors/prevention & control , Middle Aged , Neck/blood supply , Neck/innervation , Spine/innervation
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