Mariko Taniguchi

Cruciform DNA Structure Underlies the Etiology for Palindrome-mediated Human Chromosomal Translocations

There is accumulating evidence to suggest that palindromic AT-rich repeats (PATRRs) represent hot spots of double-strand breakage that lead to recurrent chromosomal translocations in humans. As a mechanism for such rearrangements, we proposed that the PATRR forms a cruciform structure that is the source of genomic instability. To test this hypothesis, we have investigated the tertiary structure of a cloned PATRR. We have observed that a plasmid containing this PATRR undergoes a conformational change, causing temperature-dependent mobility changes upon agarose gel electrophoresis. The mobility shift is observed in physiologic salt concentrations and is most prominent when the plasmid DNA is incubated at room temperature prior to electrophoresis. Analysis using two-dimensional gel electrophoresis indicates that the mobility shift results from the formation of a cruciform structure. S1 nuclease and T7 endonuclease both cut the plasmid into a linear form, also suggesting cruciform formation. Furthermore, anti-cruciform DNA antibody reduces the electrophoretic mobility of the PATRR-containing fragment. Finally, we have directly visualized cruciform extrusions from the plasmid DNA with the size expected of hairpin arms using atomic force microscopy. Our data imply that for human chromosomes, translocation susceptibility is mediated by PATRRs and likely results from their unstable conformation.

Protective Mechanisms against Pulmonary Infection with Influenza Virus. I. Relative Contribution of Polymorphonuclear Leukocytes and of Alveolar Macrophages to Protection during the Early Phase of Intranasal Infection

The relative contribution of polymorphonuclear leukocytes and macrophages in the early protection against intranasal infection of mice with influenza virus was investigated. Virus multiplication in the lung in the early phase of infection with less than 1.5 X 10(3) plaque-forming units was enhanced by X-ray irradiation. The intranasal administration of carrageenan did not influence the titre of virus. However, when mice were infected with 1.5 X 10(4) plaque-forming units, the virus titre was elevated by intranasal administration of carrageenan as well as by X-ray irradiation, but not by intraperitoneal administration of carrageenan. The intranasal administration of carrageenan not only inhibited the phagocytic activity of alveolar macrophages but also enhanced susceptibility to the virus. On the other hand, polymorphonuclear leukocytes were capable of phagocytosing the virus in vitro and were non-permissive for virus infection. Neutralizing antibody and interferon were not detectable in the early stage of the infection. These results suggested that polymorphonuclear leukocytes (X-ray-sensitive, carrageenan-resistant) were the cells primarily responsible for early protection in influenza virus infection and that after infection with a high dose of the virus alveolar macrophages (X-ray-resistant, carrageenan-sensitive) also played a protective role in the early phase.

Basement membrane fragility underlies embryonic lethality in fukutin-null mice

Fukuyama-type congenital muscular dystrophy (FCMD), associated with brain malformation due to defects in neuronal migration, is caused by mutations in fukutin. Several lines of evidence suggest that the fukutin protein plays a pivotal role in synthesis of O-mannosyl sugar moieties of alpha-dystroglycan, a cell surface laminin receptor. Here, through targeted disruption of the orthologous mouse fukutin gene, we show that the fukutin protein is essential, as homozygous-null embryos die by E9.5 of gestation. Fukutin-null embryos show phenotypic diversity, features of which include growth retardation, folding of the egg cylinder, leakage of maternal red blood cells into the yolk sac cavity, and an increased number of apoptotic cells in the ectoderm. Loss of immunoreactivity against sugar moieties in alpha-dystroglycan suggests a reduced laminin-binding capacity. Ultrastructural analysis shows thin and breached basement membranes (BMs). BM fragility may underlie all of these abnormal phenotypes, and maintenance of BM function may require fukutin-mediated glycosylation of alpha-dystroglycan early in embryonic development.

Chromosomal instability mediated by non-B DNA: Cruciform conformation and not DNA sequence is responsible for recurrent translocation in humans

Chromosomal aberrations have been thought to be random events. However, recent findings introduce a new paradigm in which certain DNA segments have the potential to adopt unusual conformations that lead to genomic instability and nonrandom chromosomal rearrangement. One of the best-studied examples is the palindromic AT-rich repeat (PATRR), which induces recurrent constitutional translocations in humans. Here, we established a plasmid-based model that promotes frequent intermolecular rearrangements between two PATRRs in HEK293 cells. In this model system, the proportion of PATRR plasmid that extrudes a cruciform structure correlates to the levels of rearrangement. Our data suggest that PATRR-mediated translocations are attributable to unusual DNA conformations that confer a common pathway for chromosomal rearrangements in humans.

Residual laminin-binding activity and enhanced dystroglycan glycosylation by LARGE in novel model mice to dystroglycanopathy

Hypoglycosylation and reduced laminin-binding activity of α-dystroglycan are common characteristics of dystroglycanopathy, which is a group of congenital and limb-girdle muscular dystrophies. Fukuyama-type congenital muscular dystrophy (FCMD), caused by a mutation in the fukutin gene, is a severe form of dystroglycanopathy. A retrotransposal insertion in fukutin is seen in almost all cases of FCMD. To better understand the molecular pathogenesis of dystroglycanopathies and to explore therapeutic strategies, we generated knock-in mice carrying the retrotransposal insertion in the mouse fukutin ortholog. Knock-in mice exhibited hypoglycosylated α-dystroglycan; however, no signs of muscular dystrophy were observed. More sensitive methods detected minor levels of intact α-dystroglycan, and solid-phase assays determined laminin binding levels to be ∼50% of normal. In contrast, intact α-dystroglycan is undetectable in the dystrophic Largemyd mouse, and laminin-binding activity is markedly reduced. These data indicate that a small amount of intact α-dystroglycan is sufficient to maintain muscle cell integrity in knock-in mice, suggesting that the treatment of dystroglycanopathies might not require the full recovery of glycosylation. To examine whether glycosylation defects can be restored in vivo, we performed mouse gene transfer experiments. Transfer of fukutin into knock-in mice restored glycosylation of α-dystroglycan. In addition, transfer of LARGE produced laminin-binding forms of α-dystroglycan in both knock-in mice and the POMGnT1 mutant mouse, which is another model of dystroglycanopathy. Overall, these data suggest that even partial restoration of α-dystroglycan glycosylation and laminin-binding activity by replacing or augmenting glycosylation-related genes might effectively deter dystroglycanopathy progression and thus provide therapeutic benefits.

Fukuyama-type congenital muscular dystrophy (FCMD) and α-dystroglycanopathy

ABSTRACT  Fukuyama‐type congenital muscular dystrophy (FCMD), Walker‐Warburg syndrome (WWS), and muscle‐eye‐brain (MEB) disease are clinically similar autosomal recessive disorders characterized by congenital muscular dystrophy, lissencephaly, and eye anomalies. Through positional cloning, we identified the gene for FCMD and MEB, which encodes the fukutin protein and the protein O‐linked mannose β1, 2‐N‐acetylgIucosaminy ltransferase (POMGnT1), respectively. Recent studies have revealed that posttranslational modification of α‐dystro‐glycan is associated with these congenital muscular dystrophies with brain malformations. In this review Fukuyama‐type congenital muscular dystrophy (FCMD), other CMDs with brain malformations, and their relation with α‐dystroglycan are discussed.

Mechanism of Protection During the Early Phase of a Generalized Viral Infection. II. Contribution of Polymorphonuclear Leukocytes to Protection against Intravenous Infection with Influenza Virus

The contribution of phagocytes to the early protection of mice inoculated intravenously with influenza virus was investigated in phagocyte-depleted mice. Following the inoculation of a sublethal dose of influenza virus, virus titres in the liver and lung of both untreated and carrageenan-treated mice were reduced rapidly by day 1 and decreased slowly to reach an undetectable level by day 7. The titres in gamma-irradiated mice decreased transiently by day 1 and increased progressively thereafter to kill all of the hosts by day 8. The clearance of virus from blood at the early stage of infection was retarded by gamma-irradiation but not by carrageenan treatment. In addition, increase in virus titres in the liver and lung in the early stage of the infection was prevented by adoptive transfer with syngeneic polymorphonuclear leukocytes into gamma-irradiated mice. No significant rise of neutralizing antibody was detectable by day 3 after the inoculation, in any of the three groups of mice. These observations imply that gamma-sensitive and carrageenan-resistant polymorphonuclear leukocytes play a protective role at the early stage in the infection, whereas fixed macrophages or natural killer cells, both of which are carrageenan-sensitive and gamma-resistant, scarcely participate in the early phase.

Screening of genes involved in chromosome segregation during meiosis I: toward the identification of genes responsible for infertility in humans.

Prophase I of male meiosis during early spermatogenesis involves dynamic chromosome segregation processes, including synapsis, meiotic recombination and cohesion. Genetic defects in the genes that participate in these processes consistently cause reproduction failure in mice. To identify candidate genes responsible for infertility in humans, we performed gene expression profiling of mouse spermatogenic cells undergoing meiotic prophase I. Cell fractions enriched in spermatogonia, leptotene/zygotene spermatocytes or pachytene spermatocytes from developing mouse testis were separately isolated by density gradient sedimentation and subjected to microarray analysis. A total of 726 genes were identified that were upregulated in leptotene/zygotene spermatocytes. To evaluate the screening efficiency for meiosis-specific genes, we randomly selected 12 genes from this gene set and characterized each gene product using reverse transcription (RT)-PCR of RNA from gonadal tissues, in situ hybridization on testicular tissue sections and subcellular localization analysis of the encoded protein. Four of the 12 genes were confirmed as genes expressed in meiotic stage and 2 of these 4 genes were novel, previously uncharacterized genes. Among the three confirmation methods that were used, RT-PCR appeared to be the most efficient method for further screening. These 726 candidates for human infertility genes might serve as a useful resource for next-generation sequencing combined with exon capture by microarray.

A fundamental study for quantitative measurement of ultrasound contrast concentration by low mechanical index contrast ultrasonography

PurposeIn high mechanical index (MI) contrast ultrasonography it has been shown that the power of contrast signal intensity (CI) has a strong linear correlation with the concentration of the ultrasound contrast agent under conditions of constant applied acoustic pressure. However, it is unclear whether the linearity is preserved in low-MI contrast ultrasonography. Thus, we investigated the relationship between ultrasound contrast concentration and CI in vitro.MethodsSolutions of the ultrasound contrast agents Definity and Imagent were prepared at concentrations of 0.5, 2, 8, 32, and 128 μl/l. Placing a jelly block between the transducer and the solution, the solutions were transmitted using pulse subtraction imaging with an MI of 0.05, 0.1, and 0.5. CI was measured in dB in a region of interest 3 mm in height placed just below the border between the jelly and the solution. Data were plotted using double logarithm scales, where the concentration was expressed in dB as 10 × log (concentration).ResultsCI in dB had a strong linear correlation with concentration in dB for both agents with any MI. Best fitted slopes were close to 1, indicating that the power of CI is proportional to the concentration.ConclusionsIn low-MI contrast ultrasonography, the power of CI is proportional to contrast concentration, and CI in dB is logarithmic to the concentration. Thus, the microbubble concentration can be quantitatively measured even in low-MI contrast ultrasonography.

A Fundamental Study for Quantitative Measurement of Ultrasound Contrast Concentration by Low Mechanical Index Contrast Ultrasonography

目的 : 高音圧コントラストエコー法において, 局所の入射音圧が一定である条件下では造影剤濃度とコントラスト強度 (CI) のパワー値の間に良好な線形性があることが分かってきた. しかし, 低音圧コントラストエコー法でも同様の線形性が保たれるか否かは不明である. そこで, 低音圧コントラストエコー法における造影剤濃度とCIとの関係を確認するためにin vitro実験を行った. 方法 : 造影剤はDefinity®とImagent®の2種類を用いた. 0.5, 2, 8, 32, 128μL/Lの5濃度の溶液を用意し, mechanical index (MI) 0.05, 0.1, 0.5のパルスサブトラクションイメージングで, ゼリーを介して溶液を撮像した. 画像上の溶液の水面直下に厚さ3mmの関心領域を置き, CIをdBで計測した. 濃度は0.5μL/Lを基準値として10×log (濃度) でdB表示し, 両側対数グラフ上で直線回帰分析を行った. 結果 : 両造影剤とも, いずれのMIにおいても, 造影剤濃度とCIとはきわめて良好な直線相関を示し, 回帰直線の傾きはCIのパワー値が造影剤濃度に比例する場合の理論値1に近かった. 結語 : 低音圧コントラストエコー法においても, CIのパワー値が造影剤濃度に比例し, CIのdB値は濃度に対して対数関係にある. 低音圧コントラストエコー法で気泡密度の定量計測が可能である.