In Vitro Studies to Define the Cell-Surface and Intracellular Targets of Polyarginine-Conjugated Sodium Borocaptate as a Potential Delivery Agent for Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) requires pharmaceutical innovations and molecular-based evidence of effectiveness to become a standard cancer therapeutic in the future. Recently, in Japan, 4-borono-L-phenylalanine (BPA) was approved as a boron agent for BNCT against head and neck (H&N) cancers. H&N cancer appears to be a suitable target for BPA-BNCT, because the expression levels of L-type amino acid transporter 1 (LAT1), one of the amino acid transporters responsible for BPA uptake, are elevated in most cases of H&N cancer. However, in other types of cancer including malignant brain tumors, LAT1 is not always highly expressed. To expand the possibility of BNCT for these cases, we previously developed poly-arginine peptide (polyR)-conjugated mercaptoundecahydrododecaborate (BSH). PolyR confers the cell membrane permeability and tumor selectivity of BSH. However, the molecular determinants for the properties are not fully understood. In this present study, we have identified the cluster of differentiation 44 (CD44) protein and translational machinery proteins as a major cell surface target and intracellular targets of BSH-polyR, respectively. CD44, also known as a stem cell-associated maker in various types of cancer, is required for the cellular uptake of polyR-conjugated molecules. We showed that BSH-polyR was predominantly delivered to a CD44High cell population of cancer cells. Once delivered, BSH-polyR interacted with the translational machinery components, including the initiation factors, termination factors, and poly(A)-biding protein (PABP). As a proof of principle, we performed BSH-polyR-based BNCT against glioma stem-like cells and revealed that BSH-polyR successfully induced BNCT-dependent cell death specifically in CD44High cells. Bioinformatics analysis indicated that BSH-polyR would be suitable for certain types of malignant tumors. Our results shed light on the biochemical properties of BSH-polyR, which may further contribute to the therapeutic optimization of BSH-BNCT in the future.

The accelerator-based boron neutron capture reaction evaluation system for head and neck cancer.

The purpose of this study is to assess accelerator-based boron neutron capture reaction (BNCR) in human tumor cell lines by colony formation assay and modified high density survival assay (HDS assay). The results of post irradiation survival rate in human oral squamous cell carcinoma and osteosarcoma using both assays were similar. Therefore, HDS assay would be efficient to evaluate BNCR in not only tumor cells but also in normal cells as BNCT screening.

A molecular epidemiological survey of Babesia, Hepatozoon, Ehrlichia and Anaplasma infections of dogs in Japan.

Tick-borne diseases are often encountered in canine clinical practice. In the present study, a molecular epidemiological survey of dogs in Japan was conducted to understand the prevalence and geographical distribution of Babesia spp., Hepatozoon spp., Ehrlichia spp. and Anaplasma spp. Pathogen-derived DNA in blood samples obtained from 722 dogs with a history of exposure to ticks and/or fleas was examined by PCR. The prevalence of Babesia gibsoni, Babesia odocoilei-like species, Hepatozoon canis and Ehrlichia spp./Anaplasma spp. was 2.4% (16/722), 0.1% (1/722), 2.5% (18/722) and 1.5% (11/722), respectively. While B. gibsoni and Ehrlichia spp./Anaplasma spp. were detected in the western part of Japan, H. canis was detected in Tohoku area in addition to western and central parts of Japan.

A nationwide survey of ixodid tick species recovered from domestic dogs and cats in Japan in 2011.

A nationwide survey of ixodid ticks was performed in 2011, during which a total of 4237 and 298 ticks were recovered from 1162 dogs and 136 cats, respectively. Haemaphysalis longicornis was the most frequently found tick species on canine hosts (739 dogs), followed by H. flava (166), Ixodes ovatus (139), and Rhipicephalus sanguineus sensu lato (70). H. hystricis, H. japonica, H. megaspinosa, H. formosensis, H. campanulata, H. ias, I. nipponensis, I. persulcatus, and Amblyomma testudinarium were also recovered. H. longicornis was also the most frequently found species on feline hosts (52 cats), followed by I. ovatus (34), A. testudinarium (19), and H. flava (12). H. hystricis, H. japonica, H. megaspinosa, I. nipponensis, I. persulcatus, I. granulatus and R. sanguineus sensu lato were also recovered from cats. The three major species of ticks found on dogs and cats, H. longicornis, H. flava, and I. ovatus, displayed a wide geographical distribution, with specimens found throughout northern and southern Japan. R. sanguineus sensu lato was primarily recovered in Okinawa, but was also found in Kanagawa, Wakayama, Hiroshima, and Yamaguchi Prefectures. A. testudinarium was mainly distributed throughout western Japan, but small numbers were also recovered from Gumma and Shizuoka Prefectures. H. longicornis was more frequently found on dogs in rural areas than those in urban or suburban areas. Exposure to woodland environments was significantly associated with H. flava and I. ovatus in dogs. Dogs in urban or suburban areas encountered R. sanguineus sensu lato more often than other tick species. Most of the cats surveyed in the present study were from rural areas. In the present study, H. hystricis and R. sanguineus sensu lato were found on cats for the first time in Japan.

Molecular survey of Babesia gibsoni using Haemaphysalis longicornis collected from dogs and cats in Japan.

A nationwide survey of Babesia gibsoni using Haemaphysalis longicornis collected from dogs and cats in Japan was conducted using molecular methods. A total of 1,341 H. longicornis, including 305 females, 14 males, 332 nymphs and 690 larvae (153 pools) from 44 prefectures, were examined by B. gibsoni-targeted PCR. Partial sequence analysis revealed that 12 of 13 positive samples sequenced, including samples from Tottori, Hiroshima, Yamaguchi, Tokushima, Ehime and Oita prefectures (all in western Japan), were identical to B. gibsoni, and 1 sample from Kyoto Prefecture was most closely related to a Babesia species recently detected from feral raccoons in Hokkaido. H. longicornis is a candidate for transmission vector tick of the new Babesia species.

Prevalence of Dirofilaria immitis among shelter dogs in Tokyo, Japan, after a decade: comparison of 1999-2001 and 2009-2011.

Changes in the seroprevalence of Dirofilaria immitis infection among shelter dogs between a decade ago and the present were evaluated. Serum samples were collected from 200 adult dogs in urban and suburban areas in Tokyo, Japan, during two 2-year periods (April 1999 to March 2001 and April 2009 to March 2011). Sera were tested for the presence of D. immitis antigen using a specific commercialized kit. The seroprevalence of D. immitis infection was 46% in 1999-2001 and 23% in 2009-2011. A decrease was observed in the prevalence of infection between 1999-2001 and 2009-2011; in particular, the prevalence in urban areas decreased significantly compared with that in suburban areas (P < 0.01). There was no significant difference in prevalence between the sexes in each period, but there was a significant difference between mixed-breed and purebred dogs (P < 0.01). The decrease in prevalence of canine heartworm disease in urban areas could be related to better veterinary care.

Microplate Agglutination Test for Canine Brucellosis Using Recombinant Antigen-Coated Beads.

Brucella canis, a facultative intracellular pathogen, is the causative agent of canine brucellosis. The diagnosis of canine brucellosis is based on bacteriological examination and serological methods, including agglutination and gel diffusion tests. In this study, four recombinant antigens, heat shock protein 60, rhizopine-binding protein, Cu-Zn superoxide dismutase, and hypothetical protein (Ag 4), were constructed. These antigens were coated on latex beads and their usefulness in the serological diagnosis of canine brucellosis was examined. All recombinant antigens showed specific reaction with sera from B. canis-infected dogs in Western blotting. In a microplate agglutination test, mixing sera from B. canis-infected dogs, but not sera from B. canis-free dogs, with single or multiple antigens-coated latex beads produced clear agglutination. Moreover, the antigen-coated latex beads did not show nonspecific agglutination in hemolyzed serum samples. A survey of canine serum samples conducted by the microplate agglutination test using single antigen-coated latex beads showed that this method would be useful in the serological diagnosis of canine brucellosis. Further investigations using more serum samples are required to confirm the usefulness of our method.

Molecular survey of Rickettsia, Ehrlichia, and Anaplasma infection of domestic cats in Japan.

The prevalence of Rickettsia, Ehrlichia, and Anaplasma in 1764 DNA samples extracted from feline peripheral blood from all 47 prefectures in Japan was evaluated by screening real-time PCR, genus-specific PCR, and DNA nucleotide sequencing. The survey revealed that all cats were negative for Rickettsia infection. Two cats were positive for Ehrlichia or Anaplasma based on the screening PCR assay. Nucleotide sequence analysis of the partial 16S rRNA including the divergent region near the 3'-end revealed that the 2 positives were most similar to Anaplasma bovis with percent identities of 99.8% and 99.2%. This was the first detection of A. bovis DNA fragments in cats. Although these 2 cats showed stomatitis, both were also infected with feline immunodeficiency virus. The relationship between A. bovis carriage and clinical disease is not yet understood.

Detection of Anaplasma bovis DNA in the peripheral blood of domestic dogs in Japan.

The prevalence of Ehrlichia and Anaplasma in 1,427 dogs from 32 Japanese prefectures was evaluated by PCR and DNA nucleotide sequencing. PCR screening demonstrated that 18 dogs (1.3%) were positive for Anaplasmataceae. Sequence analysis revealed that 14 of the amplicons were most closely related to Wolbachia spp., symbionts of Dirofilaria immitis, whereas three were identified as Anaplasma bovis. The remaining amplicon could not be sequenced. Almost the entire sequence of 16S rRNA (1,452 bp) from one of the positive specimens was determined, and subsequent phylogenetic analysis confirmed that the detected sequence was that of A. bovis. This is the first detection of A. bovis DNA fragments in dogs. Species-specific nested PCR showed that 15 (1.1%) of the 1,427 dogs involved in this study were positive for A. bovis. The geographical distribution of these dogs ranged from Aomori Prefecture in northern Japan to Kagoshima Prefecture in the south. The relationship between A. bovis infection and clinical disease is not yet clearly understood.

Development of a method of diagnosing ovarian disorders in sows and gilts using uterine ultrasonography.

In ultrasonographic diagnosis of ovarian disorders and the estrous cycle in sows, transverse observation of the uterus yielded more characteristic findings than observation of sagittal sections. Transverse ultrasonography revealed that the low progesterone (P) type uterus showed a round structure, while the high P type uterus showed a flattened structure. These results corresponded well with rectal palpation findings: the low P type uterus had a hard, pipe-like structure and the high P type a soft, balloon-like structure. For gilts, we employed a minimum convex type transrectal prostate probe that had an approximately 18 cm insertion handle. The images of the uterus obtained thereby were a similar to those obtained from sows. The above results suggest that it should be possible to diagnose and treat many ovarian disorders in sows and gilts based only on the ultrasonograhic findings. In short, ultrasonograhic findings of a round structure of the uterine wall might be an indication for PMSG (pregnant mare serum gonadotropin) treatment, while findings of a flattened structure might be an indication for PGF(2) (alpha) administration.

Three-dimensional distribution patterns of PRL, GH and ACTH cells in the house musk shrew ( Suncus murinus).

Despite a multitude of reports on the classification and distribution of anterior pituitary cells, no previous study has attempted to obtain the three-dimensional (3D) and computer-graphic distribution pattern of each cell type in the whole pituitary. Therefore, we mapped the anterior pituitary cells of the house musk shrew ( Suncus murinus) and found a distinct cellular distribution pattern. Serial horizontal sections of whole shrew pituitaries were stained immunohistochemically for prolactin (PRL), growth hormone (GH), and adrenocorticotropic hormone (ACTH) cells. The contours of positive cells and the anterior, intermediate, and posterior lobes in each section were digitized for 3D visualization by a volume rendering method. The reconstructed images and virtual frontal and sagittal slices were examined in detail. On the 3D reconstructed images, the PRL and GH cells had similar distribution patterns, although the former were concentrated in the dorsolateral and ventrocentral portions, and the latter in the dorsocentral portions of the anterior lobe. On both sides of the pituitary stalk, there lay portions that were conspicuous by scarcity of PRL and GH cells. ACTH cells were widely scattered throughout the whole anterior lobe, but they were very few in the above portions and the dorsocentral portions where GH cells were concentrated. No sex difference in the distribution patterns of each cell type was observed. However, PRL cells in females were more numerous than in males, whereas the opposite was true for GH and ACTH cells. We discuss the relationship between the formation of the spatial distribution patterns and anterior pituitary ontogeny.

The fate and role of openings formed in the mesentery of bullfrog tadpoles, with reference to the contour of mesothelial cells.

Using morphological techniques, histological changes of the mesentery were observed during the development of the bullfrog, Rana catesbeiana. The tadpoles of this species had many openings all over the mesentery from the duodenum through the large intestine. Most of the openings were elliptical and less than 3 × 2 mm in size. The openings became remarkably decreased in size and number with rapid narrowing of the mesentery occurring during the period of metamorphic climax, and had almost completely disappeared by the end of metamorphosis. Appearance and disappearance of the openings were closely correlated with the changes in the dimensions of the mesentery. Furthermore, in parallel with these changes in the openings, a noticeable alteration occurred in the shape of the mesothelial cells of the mesentery. In tadpoles having no mesenteric openings, the mesothelial cells had a polygonal contour, which became transformed once the openings were formed in the mesentery. The shapes of the transformed cells were classified into two types, one having many radiating cell processes and the other a very slender and spindle-shaped contour. Both types of cells eventually became transformed into a definitive type of cell exhibiting a roundish polygonal contour by the end of metamorphosis. From these findings it was concluded that the growing mesentery might, of necessity, give rise to the openings and transformation of the mesothelial cells to enable rapid lengthening and shortining of the intestinal tract to occur during the postembryonic development of anuran amphibians.