Hiroki Sakai

A single female-specific piRNA is the primary determiner of sex in the silkworm

The silkworm Bombyx mori uses a WZ sex determination system that is analogous to the one found in birds and some reptiles. In this system, males have two Z sex chromosomes, whereas females have Z and W sex chromosomes. The silkworm W chromosome has a dominant role in female determination, suggesting the existence of a dominant feminizing gene in this chromosome. However, the W chromosome is almost fully occupied by transposable element sequences, and no functional protein-coding gene has been identified so far. Female-enriched PIWI-interacting RNAs (piRNAs) are the only known transcripts that are produced from the sex-determining region of the W chromosome, but the function(s) of these piRNAs are unknown. Here we show that a W-chromosome-derived, female-specific piRNA is the feminizing factor of B. mori. This piRNA is produced from a piRNA precursor which we named Fem. Fem sequences were arranged in tandem in the sex-determining region of the W chromosome. Inhibition of Fem-derived piRNA-mediated signalling in female embryos led to the production of the male-specific splice variants of B. mori doublesex (Bmdsx), a gene which acts at the downstream end of the sex differentiation cascade. A target gene of Fem-derived piRNA was identified on the Z chromosome of B. mori. This gene, which we named Masc, encoded a CCCH-type zinc finger protein. We show that the silencing of Masc messenger RNA by Fem piRNA is required for the production of female-specific isoforms of Bmdsx in female embryos, and that Masc protein controls both dosage compensation and masculinization in male embryos. Our study characterizes a single small RNA that is responsible for primary sex determination in the WZ sex determination system.

Expression of Vascular Endothelial Growth Factor, Basic Fibroblast Growth Factor, and Their Receptors (Flt-1, Flk-1, and Flg-1) in Canine Vascular Tumors

Expression of vascular endothelial growth factor (VEGF), its receptors (flt-1 and flk-1), and basic fibroblast growth factor (bFGF) in canine hemangiosarcoma (HSA) and hemangiomas was investigated by immunohistochemical analysis. In addition, expression of the mRNAs of VEGF, flt-1, flk-1, and flg-1 (a receptor for bFGF), was analyzed by reverse transcriptase polymerase chain reaction (RT-PCR) and in situ hybridization (ISH) with cRNA probes. VEGF, bFGF, flt-1, and flk-1 were immunohistochemically detected in the neoplastic cells in HSAs; the staining intensity was stronger in HSAs than in hemangiomas. On the other hand, the neoplastic cells in hemangiomas exhibited very weak or no expression of VEGF, although they showed moderate expression of flt-1 and flk-1. The mRNAs of VEGF, flt-1, flk-1, and flg-1 were detected in the neoplastic cells in HSAs by ISH and RT-PCR. However, VEGF mRNA was not detectable in the neoplastic cells in hemangiomas by ISH, although it was detected in the inflammatory cells in the tumors by RT-PCR. Moreover, the HSAs that showed intense staining for flk-1 had a high proliferative activity, which was reflected as a high Ki-67 positive index. These results suggest that the expression of the growth factors and their receptors, especially flk-1, might be associated with the malignant proliferation of HSAs.

Neuropathological study of gazelle herpesvirus 1 (equine herpesvirus 9) infection in Thomson's gazelles (Gazella thomsoni)

Gazelle herpesvirus (GHV-1), correctly designated as equine herpesvirus 9, is a new type of equine herpesvirus immunologically related to equine herpesvirus 1 (EHV-1). As a sequel to a virological study, the neuropathology of encephalitis caused by GHV-1 in Thomsons gazelles (Gazella thomsoni) was examined. Seven gazelles died with or without neurological symptoms between early September and mid-October in 1993. No gross abnormalities were observed at necropsy, but all animals had non-suppurative encephalitis, characterized by necrosis and degeneration of neurons, glial reactions and perivascular cuffing in the cerebrum. Five cases showed intranuclear inclusion bodies, with the appearance of herpesvirus in the degenerating neurons. Immunohistochemically, all seven animals showed a positive reaction to EHV-1 antigen in neurons in the necrotic areas of the cortex. The clinical course and morphological features of GHV-1 encephalitis were distinct from those of EHV-1-induced encephalitis in the horse, which is characterized by vasculitis, thrombosis, ischaemia, and lack of intranuclear inclusions in neurons.

Experimental Infection of Equine Herpesvirus 9 in Dogs

Equine herpesvirus 9 (EHV-9), a new neurotropic equine herpesvirus, was inoculated intranasally at 107 plaque-forming units in five dogs to assess its pathogenicity. Dogs showed weight loss, pyrexia, anorexia, and neurologic signs on the fourth day. The EHV-9 virus was recovered from the examined brains. Histologically, dogs had a fulminant nonsuppurative encephalitis characterized by severe neuronal degeneration and loss, with intranuclear inclusions, slight glial reactions, perivascular cuffing, and multifocal hemorrhage. The olfactory bulb and the frontal and temporal lobes were predominantly affected. Immunohistochemistry revealed reactivity for EHV-9 antigen in neurons. All dogs had mild bronchopneumonia and various degrees of lymphoid necrosis. These findings indicate that dogs are fully susceptible to EHV-9 and that EHV-9 can cause fulminant encephalitis with high mortality in dogs, as in gazelles and goats.

Roles of cyclooxygenase‐2 and microsomal prostaglandin E synthase‐1 expression and β‐catenin activation in gastric carcinogenesis in N‐methyl‐N‐nitrosourea‐treated K19‐C2mE transgenic mice

K19‐C2mE transgenic (Tg) mice, simultaneously expressing cyclooxygenase‐2 (COX‐2) and microsomal prostaglandin E synthase‐1 (mPGES‐1) in the gastric mucosa under the cytokeratin 19 gene promoter, were here treated with N‐methyl‐N‐nitrosourea (MNU) and inoculated with Helicobacter pylori (H. pylori) to investigate gastric carcinogenesis. Wild‐type (WT) and Tg mice undergoing MNU treatment frequently developed tumors in the pyloric region (100% and 94.7%, respectively); multiplicity in Tg was higher than that in WT (P < 0.05) with H. pylori infection. Larger pyloric tumors were more frequently observed in Tg than in WT (P < 0.05). In addition, Tg developed fundic tumors, where WT did not. No gastric tumors were observed without MNU treatment. Transcripts of TNF‐α, iNOS, IL‐1β, and CXCL14 were up‐regulated with H. pylori infection in both genotypes and were also increased more in Tg than in WT within H. pylori‐inoculated animals. Immunohistochemical analysis demonstrated significantly greater β‐catenin accumulation in pyloric tumors, compared with those in the fundus (P < 0.01) with mutations of exon 3; 18.2% and 31.6% in MNU‐alone and MNU + H. pylori‐treated WT, whereas 21.4% and 62.5% was observed in the Tg, respectively; the latter significantly higher (P < 0.05), suggesting the role of H. pylori in Wnt activation. In conclusion, K19‐C2mE mice promoted gastric cancer in both fundic and pyloric regions. Furthermore β‐catenin activation may play the important role of pyloric carcinogenesis especially in H. pylori‐infected Tg. Induction of various inflammatory cytokines in addition to overexpression of COX‐2/mPGES‐1 could be risk factors of gastric carcinogenesis and may serve as a better gastric carcinogenesis model. (Cancer Sci 2008; 99: 2356–2364)

Ochratoxin A induces DNA double-strand breaks and large deletion mutations in the carcinogenic target site of gpt delta rats

Ochratoxin A (OTA) is a carcinogen targeting proximal tubules at the renal outer medulla (ROM) in rodents. We previously reported that OTA increased mutant frequencies of the red/gam gene (Spi(-)), primarily deletion mutations. In the present study, Spi(-) assays and mutation spectrum analyses in the Spi(-) mutants were performed using additional samples collected in our previous study. Spi(-) assay results were similar to those in our previous study, revealing large (>1kb) deletion mutations in the red/gam gene. To clarify the molecular progression from DNA damage to gene mutations, in vivo comet assays and analysis of DNA damage/repair-related mRNA and/or protein expression was performed using the ROM of gpt delta rats treated with OTA at 70, 210 or 630 µg/kg/day by gavage for 4 weeks. Western blotting and immunohistochemical staining demonstrated that OTA increased γ-H2AX expression specifically at the carcinogenic target site. In view of the results of comet assays, we suspected that OTA was capable of inducing double-strand breaks (DSBs) at the target sites. mRNA and/or protein expression levels of homologous recombination (HR) repair-related genes (Rad51, Rad18 and Brip1), but not nonhomologous end joining-related genes, were increased in response to OTA in a dose-dependent manner. Moreover, dramatic increases in the expression of genes involved in G2/M arrest (Chek1 and Wee1) and S/G2 phase (Ccna2 and Cdk1) were observed, suggesting that DSBs induced by OTA were repaired predominantly by HR repair, possibly due to OTA-specific cell cycle regulation, consequently producing large deletion mutations at the carcinogenic target site.

Expression of Myogenic Regulating Factors, Myogenin and MyoD, in Two Canine Botryoid Rhabdomyosarcomas

Myogenin and MyoD regulate the development of skeletal muscle, and their expressions are specific to the stages of myogenesis. Therefore, these myogenic regulatory proteins could be considered as sensitive and specific markers for rhabdomyosarcoma. In this report we investigated the immunohistochemical reactivities of myogenin and MyoD in two canine bladder botryoid rhabdomyosarcomas that were different in the degree of differentiation. MyoD was stained in the Ki-67 antigen-positive undifferentiated mesenchymal cells, which had proliferative activity similar to myoblasts differentiated from mesoblasts. In contrast, multinucleated neoplastic cells were positive for myogenin and α-sarcomeric actin but not for Ki-67 antigen, similar to the myotubes differentiated from myoblastic cells. The expressions of myogenin and MyoD were closely correlated to the histologic features of myogenic neoplastic cells.

Hexosaminidase-altered aberrant crypts, carrying decreased hexosaminidase α and β subunit mrnas, in colon of 1,2-dimethylhydrazine-treated rats

Aberrant crypt foci (ACF), consisting of morphologically irregular crypts, are thought to be precancerous lesions for colon cancers. For their molecular analysis, it is necessary to avoid contamination with adjacent normal crypts and stromal cells. Decreased hexosaminidase activity in ACF, which has been histochemically demonstrated, was used in the present study to classify isolated crypts in combination with morphological changes. The length, rim diameter, and width (average±SD, μm) of hexosaminidase‐positive (Hex+) crypts were 238.6±40.4, 89.5±22.9, and 57.6±14.0, respectively. For hexosaminidase‐negative (Hex‐) crypts, the values were 314.4±77.8, 140.3±45.7, and 97.3±34.7, the width being 1.69 tunes greater (P<0.0001). Crypts wider than 115 μm (approximately 2 tunes the average size of Hex+ crypts) were all from ACF, judging from hexosaminidase staining. To analyze transcription levels of Hex α and β subunits (Hexa and Hexb, respectively), real‐tune relative quantitative reverse transcription‐polymerase chain reaction (RT‐PCR) analysis was performed using the LightCycler system. In aberrant crypts, both Hexa and Hexb were significantly down‐regulated to 0.266 (P<0.002) and 0.131 (P<0.001) units, respectively, compared with those in morphologically normal crypts, with β‐actin as the internal standard. This decrease could be a molecular marker for precancerous enzyme‐altered ACF.

The class A macrophage scavenger receptor CD204 is a useful immunohistochemical marker of canine histiocytic sarcoma.

The immunohistochemical expression of the class A macrophage scavenger receptor CD204, was investigated in 50 canine histiocytic sarcomas (HSs) and compared with that of CD18, CD163, CD11d and class II molecules of the major histocompatibility complex (MHC). Expression of CD204 was also determined in 81 canine round cell tumours and pleomorphic sarcomas including T- and B-cell lymphomas, mast cell tumours, extramedullary plasmacytomas, cutaneous histiocytomas, transmissible venereal tumours, pigmented or amelanotic melanomas, poorly differentiated haemangiosarcomas and rhabdomyosarcomas. All of the 50 HSs expressed CD204, CD18 and MHC class II; 27 were positive for CD163 and seven expressed CD11d. All of the round cell tumours, except for one grade III mast cell tumour, were negative for CD204; however, they showed varying immunoreactivity patterns for CD18 and MHC class II. None of the pleomorphic sarcomas were immunoreactive for CD204. CD204 would appear to be a useful marker for canine HS.

Analysis of KIT expression and KIT exon 11 mutations in canine oral malignant melanomas

KIT, a transmembrane receptor tyrosine kinase, is one of the specific targets for anti-cancer therapy. In humans, its expression and mutations have been identified in malignant melanomas and therapies using molecular-targeted agents have been promising in these tumours. As human malignant melanoma, canine malignant melanoma is a fatal disease with metastases and the poor response has been observed with all standard protocols. In our study, KIT expression and exon 11 mutations in dogs with histologically confirmed malignant oral melanomas were evaluated. Although 20 of 39 cases were positive for KIT protein, there was no significant difference between KIT expression and overall survival. Moreover, polymerase chain reaction amplification and sequencing of KIT exon 11 in 17 samples did not detect any mutations and proved disappointing. For several reasons, however, KIT expression and mutations of various exons including exon 11 should be investigated in more cases.