The isopentenyl transferase gene is effective as a selectable marker gene for plant transformation in tobacco (Nicotiana tabacum cv. Petite Havana SRI)

 A selection method for transformed cells which does not inhibit regeneration is important for the establishment and optimization of a transformation protocol. We have assessed the 35S-ipt gene from Agrobacterium tumefaciens as a selectable marker gene. The identification of ipt-expressing cells from nontransformed cells enabled morphological selection without the use of kanamycin and also allowed for the elimination of a high proportion of nonexpressing cells. Ipt selection of tobacco leaf discs (Nicotiana tabacum cv. Petite Havana SRI) resulted in a 2.7-fold higher transformation frequency compared to kanamycin selection. Overexpression of the ipt gene favored plant regeneration from transformed cells, and the transformation frequency of the ipt plus kanamycin selection resulted in a 1.6-fold higher transformation frequency than kanamycin selection alone. These results indicate that this procedure might provide a strategy whereby transgenic plants can be efficiently obtained and some of the problems related to the use of antibiotics diminished. Received: 1 November 1999 / Revision received: 26 June 2000 / Accepted: 18 July 2000     

Immunosuppressive effects of the heat shock protein 90-binding antibiotic geldanamycin.

Ansamycin antibiotic, geldanamycin has a unique pharmacological effect to bind to heat shock protein 90 (hsp90) and deplete hsp90 substrates. We investigated the immunopharmacological effects of geldanamycin. Geldanamycin depleted cellular Raf-1 of rat splenic cells without affecting the steady state levels of hsp90 and downstream mitogen activated protein (MAP) kinases, ERK1 and ERK2. In parallel, it inhibited mitogen-induced nuclear factor-kappa B (NF-kappa B) activation in these cells. Geldanamycin also had a potent suppressive effect on recall antigen-induced T cell proliferation, with an IC50 value of 1 nM. In vivo, geldanamycin suppressed the progression of adjuvant-induced arthritis dose-dependently. These results suggest that geldanamycin exerts an immunosuppressive effect partly through destabilizing Raf-1, and raise a new strategy for the prevention of inflammatory diseases.     

Mouse lymphoma L1210 cells can be arrested in the G1 phase by adjusting cellular cysteine and glutathione

Mouse lymphoma L1210 cells (NCI line) that have low ability to take up cystine became deficient in cellular cysteine and glutathione in normal culture media. The cells entered the resting state during culture when they were seeded at high cell densities. They remained viable and were mostly present in the G1 or G0 phase. In the growth-arrested state, the cellular glutathione content was one order of magnitude lower than in the exponentially growing phase in the presence of 2-mercaptoethanol. In the arrested state, DNA synthesis was almost inhibited, and RNA and protein synthesis decreased markedly. Transfer of the cells to medium containing 2-mercaptoethanol, which improves the utilization of cystine by these cells, produced the rapid recovery of RNA and protein synthesis. DNA synthesis slowly increased, reaching a maximum after a lag period.     

Establishment and characterization of endometrial undifferentiated carcinoma cell line (TMCC-2.U)

We established new cell line designed TMCC-2.U, which suggested transformation to undifferentiated carcinoma, derived from endometrial clear cell carcinoma cell line (TMCC-2). The monolayer culture cell showed a pavement arrangement and spindle like shape. A rough-endoplasmic reticulum, mitochondria etc. are well developed. But cytoplasmic endocrine granulosa were so poorly, it suggests functional developments are poor. The TMCC-2.U cells were transplanted to nude mice which showed no typical pattern suggested undifferentiated carcinoma. Their chromosome number varied and the mode is 78. Marker chromosome were found frequency. Growth pattern and production of tumor marker are clearly differentiate from TMCC-2. As mensioned above, TMCC-2.U cell line will be very valuable in basic research on mechanism of transformation and effects of patient's serum on hystogenesity.     

A Survey of Veterinarians’ Attitudes toward Euthanasia of Companion Animals in Japan

This study investigated veterinarians’ attitudes toward euthanasia of companion animals in Japan. A nationwide survey was conducted with 932 veterinarians in small animal practices. It examined the number of times they administered euthanasia, their moral criteria for choosing euthanasia for animals, and their behavioral criteria for suggesting euthanasia to owners. According to the data analyses, on average the veterinarians administered euthanasia 2.48 times a year. For many veterinarians, two conditions were necessary to justify euthanasia for animals: “the animals are incurable and suffering” and “the owners request to euthanize the animals.” In the absence of either condition, the veterinarians were inclined to disapprove of choosing euthanasia. If the owners requested further treatment, 67% showed clear disapproval of choosing euthanasia for animals with serious medical conditions. Meanwhile, more than 76% showed clear disapproval of euthanizing healthy animals when the owners requested it. These results indicate that the owners’ request takes precedence over the animals’ condition for suffering animals, but not for healthy animals. For animals with serious medical conditions, 56% of the veterinarians answered that they would or might suggest euthanasia to the owners even though the owners requested further treatment. In this situation, for some veterinarians, the animals’ condition rather than the owners’ request might become a determinant in suggesting euthanasia to owners, even if their moral judgments were against choosing euthanasia for the animals. A decrease in the owners’ or the animals’ quality of life and the owners’ inability to pay were not primary factors in choosing or suggesting euthanasia. Having an experience of euthanizing their own animals was a key factor for the veterinarians which increased not only the number of times they administered euthanasia but also the degree of their moral approval of choosing euthanasia and their behavioral willingness to suggest it to owners.