YAP induces malignant mesothelioma cell proliferation by upregulating transcription of cell cycle-promoting genes.

Malignant mesothelioma (MM) shows frequent inactivation of the neurofibromatosis type 2 (NF2) -tumor-suppressor gene. Recent studies have documented that the Hippo signaling pathway, a downstream cascade of Merlin (a product of NF2), has a key role in organ size control and carcinogenesis by regulating cell proliferation and apoptosis. We previously reported that MMs show overexpression of Yes-associated protein (YAP) transcriptional coactivator, the main downstream effector of the Hippo signaling pathway, which results from the inactivation of NF2, LATS2 and/or SAV1 genes (the latter two encoding core components of the mammalian Hippo pathway) or amplification of YAP itself. However, the detailed roles of YAP remain unclear, especially the target genes of YAP that enhance MM cell growth and survival. Here, we demonstrated that YAP-knockdown inhibited cell motility, invasion and anchorage-independent growth as well as cell proliferation of MM cells in vitro. We analyzed genes commonly regulated by YAP in three MM cell lines with constitutive YAP-activation, and found that the major subsets of YAP-upregulating genes encode cell cycle regulators. Among them, YAP directly induced the transcription of CCND1 and FOXM1, in cooperation with TEAD transcription factor. We also found that knockdown of CCND1 and FOXM1 suppressed MM cell proliferation, although the inhibitory effects were less evident than those of YAP knockdown. These results indicate that constitutive YAP activation in MM cells promotes cell cycle progression giving more aggressive phenotypes to MM cells.

Detection of Kobe-type Babesia microti associated with Japanese human babesiosis in field rodents in central Taiwan and southeastern mainland China.

Field rodent surveys for Babesia infection were performed from 2002 to 2005 in the vicinities of human babesiosis occurrences in Taiwan and mainland China. Babesia microti was identified by microscopical examination and/or PCR in 1 Rattus coxinga and 1 Crocidura horsfieldii in central Taiwan and in 13 Niviventer confucianus and 1 Apodemus agrarius in Zhejiang and Fujian Provinces of southeastern China. Of 15 B. microti samples detected by PCR, all except 1 were shown to be the Kobe-type, the aetiological small subunit rRNA gene-type of the first Japanese patient; the exception was also a Kobe-related type. The Kobe-type had been found in rodents only in a few places including the human infection occurrence place in Japan. The internal transcribed spacer 1 to 2 sequences of the Taiwanese and Chinese Kobe-types were very similar to each other but considerably different (approx. 94% pairwise identities) from that of the Japanese Kobe-type. A Taiwanese Kobe-type strain was serologically differentiated from the Kobe strain originating from the Japanese first patient. The distribution of the Kobe-type in the vicinities of human babesiosis occurrences in Taiwan and China as well as in Japan is suggestive of involvement of the Kobe-type in Asian human babesiosis.

Lyme disease Borrelia spp. in ticks and rodents from northwestern China.

In May 1999, field surveys of Lyme disease spirochetes were conducted around the Tianshan Mountains in Xinjiang Uygur Autonomous Region in northwestern People's Republic of China. Ixodes persulcatus ticks were obtained in a Tianchi Lake valley with primary forest, while the tick fauna was poor in the semidesert or at higher altitudes in this region. Species identities were confirmed by molecular analysis in which an internal transcribed spacer sequence was used. Of 55 adult ticks, 22 (40%) were positive for spirochetes as determined by Barbour-Stoenner-Kelly culture passages. In addition, some rodents, including Apodemus uralensis (5 of 14 animals) and Cricetulus longicaudatus (the only animal examined), and some immature stages of I. persulcatus (4 of 11 ticks) that had fed on A. uralensis were positive for spirochetes. Based on 5S-23S rRNA intergenic spacer restriction fragment length polymorphism analysis and reactivity with monoclonal antibodies, 35 cultures (including double isolation cultures) were identified as Borrelia garinii (20 isolates, including 9 Eurasian pattern B isolates and 11 Asian pattern C isolates), Borrelia afzelii (10 pattern D isolates), and mixed cultures (5 cultures, including isolates that produced B. garinii patterns B and C plus B. afzelii pattern D). These findings revealed that Lyme disease pathogens are distributed in the mountainous areas in northwestern China even though it is an arid region, and they also confirmed the specific relationship between I. persulcatus and genetic patterns of Borrelia spp. on the Asian continent.

Borrelia sinica sp. nov., a lyme disease-related Borrelia species isolated in China.

A survey was performed for Lyme disease borrelia in the southern part of China, in Zhejiang, Sichuan and Anhui provinces, along the Yangtze River valley, in May of 1997 and 1998. Twenty isolates from Ixodes granulatus, Ixodes ovatus, Apodemus agrarius and Niviventer confucianus were obtained. These isolates were characterized by RFLP of the 5S-23S rDNA intergenic spacer, sequence analysis of the intergenic spacer, 16S rDNA and flagellin gene, DNA-DNA hybridization analysis, SDS-PAGE and Western blotting with mAbs. Six isolates from A. agrarius, five from I. granulatus collected in Zhejiang province and one from N. confucianus in Sichuan province were highly similar to strains 10MT and 5MT isolated in Korea and classified as Borrelia valaisiana. Four isolates from A. agrarius and I. granulatus collected in Zhejiang province generated unique RFLP patterns and phylogenetic analysis of the 16S rDNA and flagellin gene sequences suggested that the isolates should be classified as B. valaisiana. Furthermore, three isolates (CMN1a, CNM2, CMN3T) from N. confucianus captured in Sichuan province and one (CWO1) from I. ovatus in Anhui province showed lower 165 rDNA sequence similarity (less than 99.0%) to sequences of previously described Lyme disease-related Borrelia species. DNA-DNA hybridization results revealed that strains CMN3T and CMN1a were clearly distinct from all other known Lyme disease Borrelia species. Electron microscope observation showed the spirochaetes to be morphologically similar to those of Borrelia, but the cells contained only four periplasmic flagella inserted at each end of the spirochaetes. Based on these results, a new Borrelia species, Borrelia sinica sp. nov., is proposed. Strain CMN3T is the type strain of this new species.

A large outbreak of foodborne infection attributed to Providencia alcalifaciens.

The enteropathogenicity of Providencia alcalifaciens, a member of the family Enterobacteriaceae, has not yet been well established. In November 1996, a large outbreak of foodborne infection occurred in Fukui, Japan. In this study, the etiology of the outbreak was investigated. No other recognized enteropathogens were detected in patient fecal samples, but P. alcalifaciens was detected in 7 of 18 samples. The isolates were found to be clonal by pulsed-field gel electrophoresis. The patients who presented with gastroenteritis had elevated levels of specific antibody against the isolated P. alcalifaciens. The isolates showed invasion of Caco-2 cells and fluid accumulation in rabbit ileal loops. This study strongly suggests that the outbreak was caused by P. alcalifaciens. This is the first report of a large outbreak of foodborne infection attributed to the organism and provides definitive evidence that P. alcalifaciens is a causative agent of gastroenteritis.

First records of tick-borne pathogens, Borrelia, and spotted fever group Rickettsiae in Okinawajima Island, Japan.

In early April 2000, tick-borne pathogens were surveyed in the northern area of Okinawajima Island, Okinawa Prefecture, which is the southernmost area of Japan. Borrelia valaisiana, a Lyme disease spirochete, was isolated from a field mouse Mus calori, and unidentified rickettsiae of the spotted fever group were isolated from all stages of Amblyomma testudinarium. These are the first reports of these pathogens on Okinawajima Island.

Ultrastructural features of Borrelia garinii in naturally infected voles, Clethrionomys rufocanus, with special reference to the relationship with peripheral nerves.

The ultrastructural features of Borrelia garinii found in the ear tissues of the vole, Clethrionomys rufocanus, are described. The spirochetes were observed in the interstitium of connective tissue or in contact with fibroblasts and were occasionally situated in the endoneurium of the peripheral nerves. The spirochetes did not injure or enter into the fibroblasts or Schwann cells. The cytotoxicity and migration of the spirochetes are also discussed.

Prevalence of Lyme disease Borrelia spp. in ticks from migratory birds on the Japanese mainland.

Borrelia sp. prevalence in ticks on migratory birds was surveyed in central Japan. In autumn, a total of 1,733 birds representing 40 species were examined for ticks. A total of 361 ticks were obtained from 173 birds of 15 species, and these ticks were immature Haemaphysalis flava (94.4%), Haemaphysalis longicornis, Ixodes columnae, Ixodes persulcatus, Ixodes turdus, and an unidentified Ixodes species. Of these, 27 juveniles of H. flava on Turdus pallidus, Turdus cardis, or Emberiza spodocephala, 2 juveniles of I. persulcatus on T. pallidus, and 1 female H. flava molted from a T. pallidus-derived nymph were positive for the presence of Borrelia by Barbour-Stoenner-Kelly culture passages. In spring, a total of 16 ticks obtained from 102 birds of 21 species were negative for the spirochete. Isolates from 15 ticks were characterized by 5S-23S rRNA intergenic spacer restriction fragment length polymorphism analysis; all isolates were identified as Borrelia garinii with pattern B/B' based on the previous patterning. According to the intergenic spacer sequences, 2 of 15 isolates, strains Fi14f and Fi24f, were highly similar to B. garinii strains 935T of Korea and ChY13p of Inner Mongolia, China, respectively. These findings indicate that Lyme disease-causing B. garinii may have been introduced to Japan by migratory birds from northeastern China via Korea. Additionally, a case of transstadial transmission of B. garinii from nymph to adult H. flava suggests that the infected H. flava may transmit Borrelia to large animals.

Lyme disease Borrelia species in northeastern China resemble those isolated from far eastern Russia and Japan.

Fifty-nine Borrelia burgdorferi sensu lato culture isolates collected from northeastern China were characterized by 5S-23S rRNA intergenic spacer restriction fragment length polymorphism (RFLP) analysis and reactivity with monoclonal antibodies (MAbs). Among 59 culture isolates, 30 (50.8%) were Borrelia garinii and 17 (28.8%) were Borrelia afzelii, 2 were mixtures composed of B. garinii with RFLP pattern B and B. garinii with pattern C, and 9 were mixtures composed of B. garinii and B. afzelii. One isolate, ChY13p, produced a unique pattern and was identified as B. garinii based on analyses of 16S rRNA gene sequence, flagellin PCR-RFLP typing, and MAb reactivities. No Borrelia burgdorferi sensu stricto or Borrelia japonica isolates were detected. The results indicate that Lyme disease Borrelia species in northeastern China resemble those of Borrelia isolates from far eastern Russia and Japan.

Lyme disease spirochetes in ticks from northeastern China.

During May 1996, field surveys on Lyme disease spirochetes were conducted in Beijing, Shenyang, Fushun, and Inner Mongolia in northeastern China. The ticks collected consisted of 3 genera and 12 species. Of these, Ixodes persulcatus was dominant in sun-exposed vegetation in forests in Inner Mongolia; 57 Borrelia strains (55/123 unfed adults and 2/5 immature stages fed on a rodent) were obtained from this tick by BSK culture. Additionally, 2/2 Apodemus peninsulae were positive. Ixodes nipponensis, Ixodes pavlovskyi, Haemaphysalis douglasi, and Haemaphysalis megaspinosa, newly recorded in China, and other Haemaphysalis spp. were all negative for Borrelia. Based on a polymerase chain reaction restriction fragment length polymorphism analysis of the 45 strains successfully subcultured, these were classified as 29 Borrelia garinii and 16 Borrelia afelii. These strains seemed to be more closely related to Japanese strains in genetic features than to those from Europe. The result of sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested more diversity in both genospecies, but Borrelia burgdorferi sensu stricto was not found.

Location and ultrastructure of Borrelia japonica in naturally infected Ixodes ovatus and small mammals.

The internal organs of Ixodes ovatus and the ears of wild rodents (Apodemus speciosus, Eothenomys smithii) and an insectivore (Crocidura dsinezumi) were cultured to isolate borreliae; positive samples were examined for the distribution and dissemination of spirochetes in the host tissues using electron microscopy. Seven isolates were derived from the unfed ticks and the three species of mammals. These isolates were identified as Borrelia japonica judging from the outer surface protein profile using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reactivity to a B. japonica-specific monoclonal antibody. Borreliae were found only in the midgut lumen of the tick in close contact with the microvilli on the midgut epithelium; on the other hand, borreliae found in the ears of mammals existed freely in the collagenous intercellular substances of connective tissues or in close contact with fibrocytes. The ultrastructural disparities between the borreliae in ticks and mammals appeared to correspond to differences in motility. Interestingly, the borrelia which invaded through the perineurium appeared to contact the basement membrane of a Schwann cell that enclosed several nonmyelinated nerve fibers. This may offer important information regarding the involvement of the nervous system in Lyme disease.

Characterization of Borrelia spp. isolated from the tick, Ixodes tanuki and small rodents in Japan.

Spirochetes were isolated from the tick, Ixodes tanuki, as well as wood mice (Apodemus speciosus) and voles (Clethrionomys rufocanus and Eothenomys smithii), caught in Fukui, Tokushima, and Hokkaido, Japan, from 1991 to 1993. Spirochetes were characterized on the basis of protein profiles, reactivities with monoclonal antibodies (mAbs), Outer surface protein A gene (ospA) and Outer surface protein B gene (ospB) amplification analysis, rRNA gene and flagellin gene restriction fragment length polymorphism (RFLP) analysis, and DNA homology values. Protein profiles of all isolates were homologous and reacted with mAb to OspA, OspB, OspC, flagellin, and heat shock protein 60. The primer reactivity to ospA and ospB were different from those of Borrelia burgdorferi sensu stricto, B. afzelii, B. japonica, and B. garinii. Based on the DNA/DNA homology value and RFLP analysis of rRNA and flagellin gene, these Borrelia sp. isolates are a new group of B. burgdorferi sensu lato. The isolates from ticks and the host rodents were identical in these assays. We propose that these Borrelia sp. are adapted to I. tanuki and are maintained in these field rodents.

Reservoir competence of the vole, Clethrionomys rufocanus bedfordiae, for Borrelia garinii or Borrelia afzelii.

In autumn of 1994 and spring of 1995, we examined Borrelia infection among Microtinae voles, Clethrionomys rufocanus bedfordiae, in Hokkaido, Japan. In BSK culturing of the earlobe tissues of 45 C. rufocanus bedfordiae captured, twelve rodents were positive for Borrelia. Eight isolates were used for the polymerase chain reaction (PCR) and the restriction fragment length polymorphism (RFLP) analysis. According to the results, these isolates were classified into B. garinii or B. afzelii. It is considered that a common vole, C. rufocanus bedfordiae, plays a significant role in the transmission and maintenance of B. garinii and B. afzelii, similar to the role of Apodemus speciosus mice.

Identification of spirochetes isolated from wild rodents in Japan as Borrelia japonica.

Spirochetes isolated from three species of wild mice, Apodemus speciosus, Apodemus argenteus, and Eothenomys smithi, which were caught on Honshu, Japan, were characterized on the bases of protein profiles, reactivities with monoclonal antibodies (MAbs), and DNA homology values. All the isolates reacted with MAb O1441b, which is specific for Borrelia japonica, but not with MAb P62, which is specific for Borrelia burgdorferi sensu stricto, Borrelia garinii, and Borrelia afzelii. The DNA relatedness of four representatives isolates to strain HO14, a type strain of B. japonica, was 83 to 88%. From these findings, the spirochetes were identified as B. japonica.

Prevalence of antibodies to Borrelia spirochetes among wild small rodents in central and western Japan.

Field rodents serve as a reservoir for Lyme disease spirochetes. To evaluate the antibody responses of rodents against different Borrelia species in relation to fauna of vector ticks feeding on them, we examined 272 sera of wild rodents, Apodemus speciosus, A. argenteus, and Eothenomys smithii, obtained in 27 locations in central and western Japan from 1981 to 1994. As to prevalences by rodent species using immunoperoxidase test, A. speciosus, A. argenteus and E. smithii showed 29.4%, 11.6% and 30.8% reactivity to Borrelia japonica, 10.7%, 7.2% and 3.8% to B. afzelii, 0.6%, 1.4% and 0% to B. garinii, and 14.7%, 7.2% and 11.5% to an unknown Borrelia species designated as It type, respectively. Each antibody to B. japonica, B. afzelii and B. sp. It type was detected widely both in central and western Japan, but the antibody to B. garinii was scarcely detectable in any area and rodent species examined. Apodemus mice in high mountain altitudes tend to have antibody to B. afzelii or B. japonica, and those in lower altitudes tend to have B. japonica or B. sp. It type. All 13 Apodemus mice from which B. japonica or B. sp. It type were isolated showed higher titers of antibodies to each homologous Borrelia species. The present results indicate that these antibody prevalences among rodents may be associated with dominant Ixodes ovatus and sporadic I. persulcatus on the mainland of Japan, and that Apodemus mice may not be an efficient reservoir for B. garinii. Such a serosurvey is a useful measure to evaluate the natural distribution of the pathogen.

Prevalence of Lyme borrelia in ticks, especially Ixodes persulcatus (Acari: Ixodidae), in central and western Japan.

Prevalence of Lyme Borrelia in unfed ticks was surveyed in central and western Japan during spring from 1991 to 1993. The tick fauna was diverse; ticks obtained consisted of four genera and more than 12 identified species, and both southern and northern species coexist with altitudinal separation. Midguts of ticks were cultured in BSK medium for spirochetal isolation. Borrelia-positive rates for adults of both a common species, Ixodes ovatus Neumann, and a northern species, Ixodes persulcatus Schulze, in the extreme western part of the Japanese central mountainous zone (Fukui and Gifu prefectures) were 18.1 and 12.9%, respectively. A Borrelia-positive nymph of Haemaphysalis flava Neumann also was found for the first time in Japan. In contrast, I. persulcatus in southwestern Japan was localized in mountains with elevations over 800 m and was determined negative for Borrelia, whereas I. ovatus was highly positive (28.8%). These Borrelia isolates were diverse in typing of protein profiles. Our results reveal that I. persulcatus-borne Borrelia derived from northern Japan is kept within the limits of the western extremity of the central mountainous zone.

Evaluation of skin damage of cyclic monoterpenes, percutaneous absorption enhancers, by using culture human skin cells.

The cytotoxicity of monoterpenes, percutaneous absorption enhancers, to cultured human skin cells was investigated in order to quantitatively estimate their skin damage. A neutral red bioassay with epidermal keratinocytes and a contraction test of collagen gel in which dermal fibroblasts were cultured were employed for evaluating the cytotoxicity of terpenes. In the neutral red bioassay, keratinocyte proliferation was inhibited on the addition of terpenes, and cell survival remarkably decreased with an increase in the concentration of terpenes fed into the culture well. When the fibroblasts were cultured in a collagen gel matrix, the lattice of collagen contracted as the cells grew. Therefore, the application of cytotoxic agents brings about an inhibition of collagen gel contraction induced by the fibroblasts. Strong inhibition was observed in the cases of hydrocarbons in terpenes, and the inhibition was dependent on the concentration of these compounds added in the culture medium. The cytotoxicity of terpenes was compared with the skin damage evoked by the application of terpenes in rats in vivo. As a result, it was considered that the skin irritation caused by terpenes was predictable to a certain extent by means of the cytotoxic study of cultured human skin cells.

Preliminary observations on ultrastructure of borreliae in tissues of Ixodes persulcatus.

We observed Lyme borrelia by electron microscopy in the tissues of the ticks, Ixodes persulcatus, which were indicated positive for borreliae by BSK cultures of their internal organs. Borreliae (0.25 micron in diameter) were found only in the lumen of the midgut. They were closely associated with the microvilli on the midgut epithelium but never penetrated into the epithelial cells. Ultrastructural features common to Lyme borreliae., i.e., the three-layered membranes surrounding the cytoplasm and orientation of the flagella insertions, were obviously confirmed. The present results are useful to understand tick tissue-borrelia interface.