Rapid HER2 cytologic fluorescence in situ hybridization for breast cancer using noncontact alternating current electric field mixing

Background: Human epidermal growth factor receptor 2-in situ hybridization (HER2-ISH) is widely approved for diagnostic, prognostic biomarker testing of formalin- fixed paraffin-embedded tissue blocks. However, cytologic ISH analysis has a potential advantage in tumor samples such as pleural effusion and ascites that are difficult to obtain the histological specimens. Our aim was to evaluate the clinical reliability of a novel rapid cytologic HER2 fluorescence ISH protocol (rapid-CytoFISH). Materials and Methods: Using a new device, we applied a high-voltage/frequency, noncontact alternating current electric field to tissue imprints and needle rinses, which mixed the probe within microdroplets as the voltage was switched on and off (AC mixing). Cytologic samples (n = 143) were collected from patients with immunohistochemically identified HER2 breast cancers. The specimens were then tested using standard dual-color ISH using formalin-fixed paraffin-embedded tissue (FFPE-tissue DISH) for HER2-targeted therapies, CytoFISH, and rapid-CytoFISH (completed within 4 h). Results: All 143 collected cytologic specimens (50 imprinted cytology specimens from resected tumors and 93 liquid-based cytology specimens from needle rinses) were suitable for FISH analysis. The HER2/chromosome enumeration probe (CEP) 17 ratios did not significantly differ between FFPE-tissue DISH and either CytoFISH protocol. Based on HER2 scoring criteria, we found 95.1% agreement between FFPEtissue DISH and CytoFISH (Cohen\u27s kappa coefficient = 0.771 and 95% confidence interval (CI): 0.614–0.927). Conclusion: CytoFISH could potentially serve as a clinical tool for prompt determination of HER2 status in breast cancer cytology. Rapid-CytoFISH with AC mixing will enable cancer diagnoses and HER2 status to be determined on the same day a patient comes to a clinic or hospital

Genomic view of the evolution of the complement system

The recent accumulation of genomic information of many representative animals has made it possible to trace the evolution of the complement system based on the presence or absence of each complement gene in the analyzed genomes. Genome information from a few mammals, chicken, clawed frog, a few bony fish, sea squirt, fruit fly, nematoda and sea anemone indicate that bony fish and higher vertebrates share practically the same set of complement genes. This suggests that most of the gene duplications that played an essential role in establishing the mammalian complement system had occurred by the time of the teleost/mammalian divergence around 500 million years ago (MYA). Members of most complement gene families are also present in ascidians, although they do not show a one-to-one correspondence to their counterparts in higher vertebrates, indicating that the gene duplications of each gene family occurred independently in vertebrates and ascidians. The C3 and factor B genes, but probably not the other complement genes, are present in the genome of the cnidaria and some protostomes, indicating that the origin of the central part of the complement system was established more than 1,000 MYA

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead

Evolution of the complement components with unique domain structure

To elucidate the evolutionary origin and history of the blood complement system, comprehensive cloning of the five complement gene families, the complement component 3 (C3), factor B (fB), mannan-binding protein-associated serine protease (MASP), complement component 6 (C6), and factor I (fI) families, was performed in agnathan lamprey and cnidarian sea anemone. In additions, for the C6 family which was not found in these animals, chondrichthyes shark was analyzed by RT-PCR using the consensus-degenerate hybrid oligonucleotide primers. The draft genome search and RACE analysis using cnidarian, Nematostella vectensis resulted in identification of the C3, fB and MASP genes. These genes were completely absent in the draft genome sequences of placozoa, porifera, and choanofllagelata, indicating that the multi-component complement system was established in the early stage of eumetazoan evolution before the divergence of Cnidaria and Bilateria. In situ hybridization showed the endoderm-specific expression of the identified cnidarian complement genes, indicating that the cnidarian complement system acts mainly in the primitive gut cavity called coelenteron, the only cavity facing endoderm. On the other hand, the liver EST analysis of lamprey, Lethenteron japonicum, gave the genes for the C3, fB, MASP, and fI families. However, no evidence for the gene duplication/functional divergence within these families, which was essential for establishing the classical activation pathway of the mammalian complement system, was obtained. RT-PCR analysis using the universal primers for the C6 family genes identified one C6 gene from shark but none from lamprey, indicating that the unique domain structure of C6 family was established in a common ancestor of the jawed vertebrate.These results suggest that the complement system comprising at least three components, C3, fB, and MASP, was established in the common ancestor of eumetazoan animals more than 600 million years ago. Function of the primitive complement system was most probably the protection of the gut cavity with primitive circulatory function. Remaining two families, the fI and C6 families, appeared in the primitive vertebrates, before the divergence of cyclostomes for the fI family (more than 500 million years ago), and before the divergence of the Chondrichthyes for the C6 family (more than 400 million years ago), respectively. The gene duplication/functional divergence within each complement gene family, which played essential roles in establishing the sophisticated complement system of jawed vertebrates, was occurred immediately after appearance of all the five complement gene families in the common ancestor of the jawed vertebrates報告番号: 甲24505 ; 学位授与年月日: 2009-03-23 ; 学位の種別: 課程博士 ; 学位の種類: 博士(理学) ; 学位記番号: 博理第5403号 ; 研究科・専攻: 理学系研究科生物科学専

The broad distribution of GP2 in mucous glands and secretory products

GP2, a GPI-anchored glycoprotein that is a useful marker for M cells of Peyer's patches, is functionally related to the uptake of pathogenic bacteria in the gut lumen. Our immunostaining throughout the whole body of mice detected a broader localization than previously found of GP2 in various mucous glands and secretory cells. In the oral cavity, the palatine gland and lingual gland intensely expressed GP2 with immunolabeling along the basolateral membrane of acini and in luminal secretions of ducts. Secretory portions of the duodenal gland as well as the pancreas were immunoreactive for GP2 in the digestive tract. Luminal contents in the small intestine contained aggregations of GP2-immunoreactive substances which mixed with bacteria. The bulbourethral gland of Cowper displayed the GP2 immunoreactivity among the male reproductive organs. The vaginal epithelium contained many GP2-immunoreactive goblet-like cells, the occurrence of which dramatically changed according to the estrous cycle. These findings show that GP2 is a popular secretory product released from mucous glands and secretory cells and may support defense mechanisms against pathogenic bacteria in the tubular organs open to the external milieu

固有のドメイン構造を持つ補体系因子の進化

To elucidate the evolutionary origin and history of the blood complement system, comprehensive cloning of the five complement gene families, the complement component 3 (C3), factor B (fB), mannan-binding protein-associated serine protease (MASP), complement component 6 (C6), and factor I (fI) families, was performed in agnathan lamprey and cnidarian sea anemone. In additions, for the C6 family which was not found in these animals, chondrichthyes shark was analyzed by RT-PCR using the consensus-degenerate hybrid oligonucleotide primers. The draft genome search and RACE analysis using cnidarian, Nematostella vectensis resulted in identification of the C3, fB and MASP genes. These genes were completely absent in the draft genome sequences of placozoa, porifera, and choanofllagelata, indicating that the multi-component complement system was established in the early stage of eumetazoan evolution before the divergence of Cnidaria and Bilateria. In situ hybridization showed the endoderm-specific expression of the identified cnidarian complement genes, indicating that the cnidarian complement system acts mainly in the primitive gut cavity called coelenteron, the only cavity facing endoderm. On the other hand, the liver EST analysis of lamprey, Lethenteron japonicum, gave the genes for the C3, fB, MASP, and fI families. However, no evidence for the gene duplication/functional divergence within these families, which was essential for establishing the classical activation pathway of the mammalian complement system, was obtained. RT-PCR analysis using the universal primers for the C6 family genes identified one C6 gene from shark but none from lamprey, indicating that the unique domain structure of C6 family was established in a common ancestor of the jawed vertebrate.These results suggest that the complement system comprising at least three components, C3, fB, and MASP, was established in the common ancestor of eumetazoan animals more than 600 million years ago. Function of the primitive complement system was most probably the protection of the gut cavity with primitive circulatory function. Remaining two families, the fI and C6 families, appeared in the primitive vertebrates, before the divergence of cyclostomes for the fI family (more than 500 million years ago), and before the divergence of the Chondrichthyes for the C6 family (more than 400 million years ago), respectively. The gene duplication/functional divergence within each complement gene family, which played essential roles in establishing the sophisticated complement system of jawed vertebrates, was occurred immediately after appearance of all the five complement gene families in the common ancestor of the jawed vertebratesUniversity of Tokyo (東京大学

GP2-expressing cells in the conjunctiva and tear ducts of mice : identification of a novel type of cells in the squamous stratified epithelium

GP2 is a membrane-associated secretory protein originally identified in zymogen granules of pancreatic acinar cells. Recently, this glycoprotein has attracted attention as a marker substance of M cells of Peyer's patches and for its involvement in the selective uptake of pathological bacteria via M cells. When we stained the conjunctiva and tear ducts of mice using a GP2 antibody, all goblet cells in the squamous stratified epithelium of the conjunctiva were intensely immunolabeled, while goblet cells in the intestine and airway were devoid of the immunoreactivity, indicating that the conjunctiva contains a special type of goblet cell. Further immunostaining for GP-2 labeled dispersed cells of peculiar shapes within the stratified squamous epithelium in the lacrimal canaliculi, lacrimal sac, and nasolacrimal duct. The GP2-immunoreactive cells in the tear duct projected arched or branched processes toward the basement membrane. Electron-microscopically, immunogold particles for GP2 outlined the basolateral plasma membrane of both the conjuntival goblet cells and the peculiarly shaped cells in the tear duct. Intracellularly, GP2 products of the goblet cells were localized around secretory granules in the apical cytoplasm and those of the tear duct cells inside the vesicles. The luminal contents close to apical plasma membrane were heavily labeled with immunogold particles, suggesting an exocytosis-based targeting of GP2 to the plasma membrane and its release into the lumen. The possible function of GP2 in tear ducts is discussed in relation to a defense system against invasive microoranisms and antigens