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1.
Cureus ; 15(1): e33338, 2023 Jan.
Article in English | MEDLINE | ID: mdl-36741613

ABSTRACT

The survival rate in congenital diaphragmatic hernia (CDH) with complex heart defects is low. Although the current consensus on the indications for surgical repair of CDH without heart defects has improved surgical outcomes, the surgical indication for CDH with complex heart defects remains unclear. Herein, we report the perioperative management of a patient with univentricular circulation who underwent CDH repair. Thus, patients with CDH complicated by univentricular anatomy may tolerate surgery depending on preserved respiratory function.

2.
Biomech Model Mechanobiol ; 15(5): 1091-100, 2016 10.
Article in English | MEDLINE | ID: mdl-26578077

ABSTRACT

The response to the mechanical loading of bone tissue has been extensively investigated; however, precisely how much strain intensity is necessary to promote bone formation remains unclear. Combination studies utilizing histomorphometric and numerical analyses were performed using the established murine maxilla loading model to clarify the threshold of mechanical strain needed to accelerate bone formation activity. For 7 days, 191 kPa loading stimulation for 30 min/day was applied to C57BL/6J mice. Two regions of interest, the AWAY region (away from the loading site) and the NEAR region (near the loading site), were determined. The inflammatory score increased in the NEAR region, but not in the AWAY region. A strain intensity map obtained from [Formula: see text] images was superimposed onto the images of the bone formation inhibitor, sclerostin-positive cell localization. The number of sclerostin-positive cells significantly decreased after mechanical loading of more than [Formula: see text] in the AWAY region, but not in the NEAR region. The mineral apposition rate, which shows the bone formation ability of osteoblasts, was accelerated at the site of surface strain intensity, namely around [Formula: see text], but not at the site of lower surface strain intensity, which was around [Formula: see text] in the AWAY region, thus suggesting the existence of a strain intensity threshold for promoting bone formation. Taken together, our data suggest that a threshold of mechanical strain intensity for the direct activation of osteoblast function and the reduction of sclerostin exists in a murine maxilla loading model in the non-inflammatory region.


Subject(s)
Maxilla/physiology , Models, Biological , Osteoblasts/physiology , Stress, Mechanical , Adaptor Proteins, Signal Transducing , Animals , Bone Density , Cell Count , Glycoproteins/metabolism , Intercellular Signaling Peptides and Proteins , Male , Maxilla/cytology , Mice, Inbred C57BL , Osteocytes/cytology , Osteogenesis , Weight-Bearing
3.
Arthritis Res Ther ; 17: 251, 2015 Sep 12.
Article in English | MEDLINE | ID: mdl-26373710

ABSTRACT

INTRODUCTION: We designed OP3-4 (YCEIEFCYLIR), a cyclic peptide, to mimic the soluble osteoprotegerin (OPG), and was proven to bind to RANKL (receptor activator of NF-κB ligand), thereby inhibiting osteoclastogenesis. We recently found that another RANKL binding peptide, W9, could accelerate bone formation by affecting RANKL signaling in osteoblasts. We herein demonstrate the effects of OP3-4 on bone formation and bone loss in a murine model of rheumatoid arthritis. METHODS: Twenty-four seven-week-old male DBA/1J mice were used to generate a murine model of collagen-induced arthritis (CIA). Then, vehicle or OP3-4 (9 mg/kg/day or 18 mg/kg/day) was subcutaneously infused using infusion pumps for three weeks beginning seven days after the second immunization. The arthritis score was assessed, and the mice were sacrificed on day 49. Thereafter, radiographic, histological and biochemical analyses were performed. RESULTS: The OP3-4 treatment did not significantly inhibit the CIA-induced arthritis, but limited bone loss. Micro-CT images and quantitative measurements of the bone mineral density revealed that 18 mg/kg/day OP3-4 prevented the CIA-induced bone loss at both articular and periarticular sites of tibiae. As expected, OP3-4 significantly reduced the CIA-induced serum CTX levels, a marker of bone resorption. Interestingly, the bone histomorphometric analyses using undecalcified sections showed that OP3-4 prevented the CIA-induced reduction of bone formation-related parameters at the periarticular sites. CONCLUSION: The peptide that mimicked OPG prevented inflammatory bone loss by inhibiting bone resorption and stimulating bone formation. It could therefore be a useful template for the development of small molecule drugs for inflammatory bone loss.


Subject(s)
Arthritis, Experimental/drug therapy , Bone Resorption/prevention & control , Cartilage, Articular/drug effects , Oligopeptides/pharmacology , RANK Ligand/metabolism , Amino Acid Sequence , Animals , Arthritis, Experimental/metabolism , Arthritis, Experimental/pathology , Arthritis, Rheumatoid/drug therapy , Arthritis, Rheumatoid/metabolism , Arthritis, Rheumatoid/pathology , Bone Density/drug effects , Bone Resorption/metabolism , Cartilage, Articular/metabolism , Cartilage, Articular/pathology , Cell Differentiation/drug effects , Cells, Cultured , Collagen Type I/blood , Enzyme-Linked Immunosorbent Assay , Infusions, Subcutaneous , Male , Mice, Inbred DBA , Oligopeptides/administration & dosage , Oligopeptides/metabolism , Osteoblasts/drug effects , Osteoblasts/metabolism , Osteoclasts/drug effects , Osteoclasts/metabolism , Osteogenesis/drug effects , Osteoprotegerin/administration & dosage , Osteoprotegerin/metabolism , Osteoprotegerin/pharmacology , Peptides/blood , Protein Binding , Tibia/drug effects , Tibia/metabolism , Tibia/pathology , X-Ray Microtomography
4.
Genome Biol ; 16: 161, 2015 Aug 14.
Article in English | MEDLINE | ID: mdl-26268681

ABSTRACT

BACKGROUND: CTCF and BORIS (CTCFL), two paralogous mammalian proteins sharing nearly identical DNA binding domains, are thought to function in a mutually exclusive manner in DNA binding and transcriptional regulation. RESULTS: Here we show that these two proteins co-occupy a specific subset of regulatory elements consisting of clustered CTCF binding motifs (termed 2xCTSes). BORIS occupancy at 2xCTSes is largely invariant in BORIS-positive cancer cells, with the genomic pattern recapitulating the germline-specific BORIS binding to chromatin. In contrast to the single-motif CTCF target sites (1xCTSes), the 2xCTS elements are preferentially found at active promoters and enhancers, both in cancer and germ cells. 2xCTSes are also enriched in genomic regions that escape histone to protamine replacement in human and mouse sperm. Depletion of the BORIS gene leads to altered transcription of a large number of genes and the differentiation of K562 cells, while the ectopic expression of this CTCF paralog leads to specific changes in transcription in MCF7 cells. CONCLUSIONS: We discover two functionally and structurally different classes of CTCF binding regions, 2xCTSes and 1xCTSes, revealed by their predisposition to bind BORIS. We propose that 2xCTSes play key roles in the transcriptional program of cancer and germ cells.


Subject(s)
DNA-Binding Proteins/metabolism , Enhancer Elements, Genetic , Promoter Regions, Genetic , Repressor Proteins/metabolism , Animals , Binding Sites , CCCTC-Binding Factor , Cell Line , Chromatin/chemistry , DNA/chemistry , DNA/metabolism , Genome , Humans , K562 Cells , Male , Mice , Neoplasms/genetics , Nucleotide Motifs , Protein Binding , Spermatids/metabolism , Spermatozoa/metabolism , Transcription, Genetic
5.
J Histochem Cytochem ; 60(8): 611-9, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22610462

ABSTRACT

The authors established a chromogen-based, double immunolabeling method using antibodies from the same species without any unwanted cross-reactivity. In addition, time-consuming staining steps were shortened by using polymer-based secondary antibodies. Taking advantage of the nature of the chromogen 3-amino-9-ethylcarbazole (AEC), which is used as a horseradish peroxidase substrate for antibody detection, the AEC-derived signals in the first color development were easily eliminated by alcohol treatment. Therefore, the signals from the first staining did not interfere with those from the subsequent second staining, which used the chromogen 3,3'-diaminobenzidine. The co-localization of antigens within the same cell could be confirmed using this method, because cell images of the individual dye staining steps could be obtained and developed. The images from each step could be expressed in pseudo-colors in a dark field by using a computer. As a result, merged images could be constructed that resembled the images acquired by the fluorescent immunolabeling technique. The resolution of this method enabled analysis of the coexpression of two antigens in the same cell in the same section. The authors have named this staining technique the elucidation of the coexpression of two antigens in a cell using antibodies from the same species (ECSS).


Subject(s)
3,3'-Diaminobenzidine/chemistry , Antibodies/chemistry , Antigens/metabolism , Carbazoles/chemistry , Chromogenic Compounds/chemistry , Immunohistochemistry/methods , Animals , Antigens/immunology , Antigens, Nuclear/immunology , Antigens, Nuclear/metabolism , Cross Reactions , Glial Fibrillary Acidic Protein/immunology , Glial Fibrillary Acidic Protein/metabolism , Horseradish Peroxidase/chemistry , Male , Mice , Nerve Tissue Proteins/immunology , Nerve Tissue Proteins/metabolism , Rabbits , Rats , Rats, Sprague-Dawley , Spinal Cord/metabolism , Synaptophysin , Vesicular Acetylcholine Transport Proteins/immunology , Vesicular Acetylcholine Transport Proteins/metabolism , Vesicular Transport Proteins/immunology , Vesicular Transport Proteins/metabolism
6.
J Biol Chem ; 286(31): 27378-88, 2011 Aug 05.
Article in English | MEDLINE | ID: mdl-21659515

ABSTRACT

Cancer-testis antigens (CTAs) are normally expressed in testis but are aberrantly expressed in a variety of cancers with varying frequency. More than 100 proteins have been identified as CTA including testes-specific protease 50 (TSP50) and the testis-specific paralogue of CCCTC-binding factor, BORIS (brother of the regulator of imprinted sites). Because many CTAs are considered as excellent targets for tumor immunotherapy, understanding the regulatory mechanisms governing their expression is important. In this study we demonstrate that BORIS is directly responsible for the transcriptional activation of TSP50. We found two BORIS binding sites in the TSP50 promoter that are highly conserved between mouse and human. Mutations of the binding sites resulted in loss of BORIS binding and the ability of BORIS to activate the promoter. However, although expression of BORIS was essential, it was not sufficient for high expression of TSP50 in cancer cells. Further studies showed that binding of BORIS to the target sites was methylation-independent but was diminished by nucleosomal occupancy consistent with the findings that high expression of TSP50 was associated with increased DNase I sensitivity and high BORIS occupancy of the promoter. These findings indicate that BORIS-induced expression of TSP50 is governed by accessibility and binding of BORIS to the promoter. To our knowledge this is the first report of regulated expression of one CTA by another to be validated in a physiological context.


Subject(s)
DNA-Binding Proteins/physiology , Promoter Regions, Genetic , Serine Endopeptidases/genetics , Animals , Base Sequence , Binding Sites , DNA Primers , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Humans , Mice , Mice, Knockout , NIH 3T3 Cells , Polymerase Chain Reaction , Protein Binding , Sequence Homology, Nucleic Acid , Serine Endopeptidases/metabolism
7.
PLoS One ; 5(11): e13872, 2010 Nov 08.
Article in English | MEDLINE | ID: mdl-21079786

ABSTRACT

BACKGROUND: BORIS/CTCFL is a paralogue of CTCF, the major epigenetic regulator of vertebrate genomes. BORIS is normally expressed only in germ cells but is aberrantly activated in numerous cancers. While recent studies demonstrated that BORIS is a transcriptional activator of testis-specific genes, little is generally known about its biological and molecular functions. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that BORIS is expressed as 23 isoforms in germline and cancer cells. The isoforms are comprised of alternative N- and C-termini combined with varying numbers of zinc fingers (ZF) in the DNA binding domain. The patterns of BORIS isoform expression are distinct in germ and cancer cells. Isoform expression is activated by downregulation of CTCF, upregulated by reduction in CpG methylation caused by inactivation of DNMT1 or DNMT3b, and repressed by activation of p53. Studies of ectopically expressed isoforms showed that all are translated and localized to the nucleus. Using the testis-specific cerebroside sulfotransferase (CST) promoter and the IGF2/H19 imprinting control region (ICR), it was shown that binding of BORIS isoforms to DNA targets in vitro is methylation-sensitive and depends on the number and specific composition of ZF. The ability to bind target DNA and the presence of a specific long amino terminus (N258) in different isoforms are necessary and sufficient to activate CST transcription. Comparative sequence analyses revealed an evolutionary burst in mammals with strong conservation of BORIS isoproteins among primates. CONCLUSIONS: The extensive repertoire of spliced BORIS variants in humans that confer distinct DNA binding and transcriptional activation properties, and their differential patterns of expression among germ cells and neoplastic cells suggest that the gene is involved in a range of functionally important aspects of both normal gametogenesis and cancer development. In addition, a burst in isoform diversification may be evolutionarily tied to unique aspects of primate speciation.


Subject(s)
Alternative Splicing , DNA-Binding Proteins/genetics , Gametogenesis/genetics , Promoter Regions, Genetic/genetics , Amino Acid Sequence , Animals , Base Sequence , CCCTC-Binding Factor , DNA Methylation , DNA-Binding Proteins/metabolism , Female , Gene Expression Regulation , HCT116 Cells , HEK293 Cells , Humans , K562 Cells , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Male , Microscopy, Fluorescence , Molecular Sequence Data , Neoplasms/genetics , Neoplasms/pathology , Protein Isoforms/genetics , Repressor Proteins/genetics , Repressor Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , Testis/cytology , Testis/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , Zinc Fingers
8.
Mol Cell Biol ; 30(10): 2473-84, 2010 May.
Article in English | MEDLINE | ID: mdl-20231363

ABSTRACT

Previously, it was shown that the CTCF paralogous gene, BORIS (brother of the regulator of imprinted sites) is expressed in male germ cells, but its function in spermatogenesis has not been defined. To develop an understanding of the functional activities of BORIS, we generated BORIS knockout (KO) mice. Mice homozygous for the null allele had a defect in spermatogenesis that resulted in small testes associated with increased cell death. The defect was evident as early as postnatal day 21 and was manifested by delayed production of haploid cells. By gene expression profiling, we found that transcript levels for Gal3st1 (also known as cerebroside sulfotransferase [CST]), known to play a crucial role in meiosis, were dramatically reduced in BORIS KO testes. We found that CST is expressed in testis as a novel testis-specific isoform, CST form F(TS), that has a short exon 1f. We showed that BORIS bound to and activated the promoter of CST form F(TS). Mutation of the BORIS binding site in the promoter reduced the ability of BORIS to activate the promoter. These findings define transcriptional regulation of CST expression as a critical role for BORIS in spermatogenesis.


Subject(s)
DNA-Binding Proteins/metabolism , Isoenzymes/metabolism , Spermatogenesis , Sulfotransferases/metabolism , Testis/enzymology , Animals , Base Sequence , DNA-Binding Proteins/genetics , Gene Expression Regulation, Neoplastic , Gene Targeting , Humans , Isoenzymes/genetics , Male , Mice , Mice, Knockout , Molecular Sequence Data , Promoter Regions, Genetic , Spermatogenesis/physiology , Sulfotransferases/genetics
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