ABSTRACT. Theileria orientalis is one of the benign species of Theileria that is widely distributed in Japan and is sometimes responsible for serious economic losses in the livestock industry. In the present study, we surveyed the current status of T. orientalis infection in grazing cattle in the eastern areas of Hokkaido (Taiki, Otofuke, Shintoku, and Shin-Hidaka districts) using molecular methods, as well as traditional methods, of diagnosis. The genes encoding the major piroplasm surface protein (MPSP) and p23 of T. orientalis were identified using highly detectable polymerase chain reaction (PCR). Results of the MPSP-PCR assay indicated that grazing cattle in these districts, after about 1.5 months pasturage, showed high rates of infection, ranging from 10.0-64.8%. Although the main MPSP and p23 genotypes detected were the Ikeda-or Chitose-types, an MPSP gene closely relating to that found in Okinawa prefecture, and a p23 gene closely relating to the Australian (Warwick) Buffeli-type gene, were found in the cattle in Shintoku and Shin-Hidaka districts. The present survey indicated that there were at least five types of T. orientalis classified by their MPSP genes in Hokkaido, Japan, and that T. orientalis infection rates are still high in this region.

We describe the characterization of a novel Rickettsia species cultivated from Dermacentor ticks collected in Russia and France, for which we propose the name Rickettsia raoultii sp. nov. Using multigene sequencing, we demonstrated that five rickettsial isolates from Dermacentor silvarum, Dermacentor reticulatus, Dermacentor marginatus and Dermacentor nuttalli ticks were classified within this novel spotted fever rickettsia species. This rickettsia also exhibited a serotype distinct from previously described Rickettsia species. The type strain of Rickettsia raoultii sp. nov. is strain Khabarovsk T (5CSUR R3 T 5ATCC VR-1596 T ).

New therapy development is critically needed for ovarian cancer. We used the chicken egg CAM assay to evaluate efficacy of anticancer drug delivery using recently developed biodegradable PMO (periodic mesoporous organosilica) nanoparticles. Human ovarian cancer cells were transplanted onto the CAM membrane of fertilized eggs, resulting in rapid tumor formation. The tumor closely resembles cancer patient tumor and contains extracellular matrix as well as stromal cells and extensive vasculature. PMO nanoparticles loaded with doxorubicin were injected intravenously into the chicken egg resulting in elimination of the tumor. No significant damage to various organs in the chicken embryo occurred. In contrast, injection of free doxorubicin caused widespread organ damage, even when less amount was administered. The lack of toxic effect of nanoparticle loaded doxorubicin was associated with specific delivery of doxorubicin to the tumor. Furthermore, we observed excellent tumor accumulation of the nanoparticles. Lastly, a tumor could be established in the egg using tumor samples from ovarian cancer patients and that our nanoparticles were effective in eliminating the tumor. These results point to the remarkable efficacy of our nanoparticle based drug delivery system and suggests the value of the chicken egg tumor model for testing novel therapies for ovarian cancer.

Rickettsia massiliae, strain Bar29, was detected in engorged female ticks of the Rhiphicephalus sanguineus group collected in Corsica, a French Mediterranean island. Ticks were identified by molecular analysis as Rhipicephalus turanicus (Pomerantsev) (Acari: Ixodidae). Twenty larvae of the second generation obtained from a R. massiliae-infected, engorged female were tested by polymerase chain reaction (PCR) and all were positive for R. massiliae. Larvae of the same cohort were fed on rabbits and specimens of subsequent stages of the second and third generation of ticks were tested by PCR. Both transovarial and transstadial transmission were demonstrated; the transovarial transmission rate was estimated at 100%. A high filial infection rate was demonstrated; 132 out of 134 larvae obtained from five infected females of the fourth generation were infected. When saliva samples from half-engorged Rh. turanicus of the second generation were tested by PCR, four out of five were positive. Rickettsia massiliae was detected in faeces of infected ticks by PCR and immunofluorescence assay, although no rickettsiae could be maintained in culture. Co-feeding/transsexual transmission of R. massiliae Bar29 was demonstrated by feeding male Rh. turanicus on a rabbit with Rhipicephalus sanguineus (Latreille) (Acari: Ixodidae) females (the latter were the only uninfected ticks available). Infection was subsequently detected in nine out of the thirteen females (69.2%). These results suggest that Rh. turanicus ticks are potential vectors and reservoirs for R. massiliae Bar29.

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Melanocytes are pigmented cells distributed in humans in several organs like the epidermis, the leptomeninges, the eye, and the inner ear. Epidermal melanocytes, whether derived from adult or neonatal skin, proliferate well in a medium supplemented with phorbol esters and other mitogens before they undergo senescence. Potent cAMP inducers like cholera toxin are also growth promoters for neonatal melanocytes but only transient growth stimulators for cells derived from adults. We used this cellular system to delineate biochemical pathways involved in proliferation and in terminal differentiation. Here we show that after a period of 4-8 wk of sustained proliferation in the presence of cholera toxin, the adult melanocytes became round, flat, and enlarged. These changes were associated with terminal growth and preceded by a five-to sixfold increase in cAMP levels and an 8-to 10-fold increase in melanin content. The simultaneous addition of phorbol esters and cholera toxin did not prevent cells from reaching terminal differentiation. Identified targets for phorbol esters are protein kinase C (PKC) and the mitogen-activated kinases (MAPKs), also called extracellular signal-regulated kinases (ERKs). PKC was found to be similarly regulated in proliferating and in terminally differentiated melanocytes. Proliferating melanocytes in early or late passage showed identical activation of the kinase ERK2. This kinase was rapidly phosphorylated upon phorbol 12-myristate 13-acetate (PMA) addition and specifically accumulated in the nucleus of the cells, whereas in unstimulated cells it had a perinuclear distribution. In contrast, senescent and terminally differentiated cells were unable to phosphorylate tyrosine residues of the ERK2 gene product in spite of presenting normal amounts of ERK2 protein. In addition, ERK2 did not show the nuclear accumulation observed in proliferating melanocytes after PMA activation and remained localized in the perinuclear area. These results demonstrate that senescent and terminally differentiated melanocytes share a common block in a critical pathway thought to integrate multiple intracellular signals transmitted by various second messengers and specifically prevent the continuation of the signal transduction cascade initiated by PMA activation of PKC. (Lorton and Nordlund, 1991;Tassabehji et al., 1992;Urabe et al., 1993). When humans reach their fourth or fifth decade of life, the number of melanocytes in the epidermis, hair bulbs, eyes, and mucus membranes and the number of oral and cutaneous nevi begin to decrease (Nordlund, 1986). At the same time, there is a significant increase in the incidence of skin cancers. It is not known if the decrease in melanocyte numbers as humans age is because of premature senescence, terminal differentiation, or both phenomena. Possibly one result of the loss of melanocytes is a permissive environment for the development of malignancies. The possibility to grow human melanocytes in vitro to study the cellular and molecular mechanisms involved in melanocyte mig...

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