Plants transformed with a cistron of a potato virus Y protease (NIa) are resistant to virus infection.

An oligonucleotide carrying signals for translation initiation in plants was engineered upstream to a cDNA clone containing nucleotides 5812-7260 of the potato virus Y (PVY) genome. This fragment contains all but the first 100 5' terminal bases of the cistron encoding one of the PVY proteases (NIa) as well as the first 251 bases of the next cistron (NIb). Nicotiana tabacum cv. SR1 plants were transformed with this fragment. The presence of the NIa sequences in transformed plants was determined by hybridization or PCR, and its expression was ascertained by reverse transcription coupled to PCR. Plants expressing NIa were self-pollinated, and the R1 kanamycin-resistant progeny were rechecked for NIa expression. Several of these plants were found to be resistant to PVY infection, inasmuch as they did not develop symptoms for at least 50 days (the duration of the experiments), and no viral accumulation could be detected in their leaves by ELISA. All of the descendents of resistant homozygous R2 plants were also resistant. Several of the plants transformed with the last three cistrons of PVY (bases 5812-9704; NIa-NIb-coat protein) were also resistant to PVY. None of the transformed plants exhibited resistance to tobacco mosaic virus. Exposure of the plants to 35 degrees C for 48 hr prior to inoculation lowered, but did not abolish, resistance.

Expression of the "helper component" protein of potato virus Y (PVY) in E. coli: possible involvement of a third protease.

Transmission of potyviruses by aphids depends on the presence of a virus encoded helper-component protein (HC) that also exhibits protease activity. HC was expressed in E. coli from two types of clones: a full-length cDNA clone of PVY and two 5' end clones containing the first three cistrons (3.6-3.7 kbp). The clones derived from the 5' end of PVY expressed HC of the size of the mature component. Other proteins reacting with antibodies to HC were also observed, and their sizes corresponded with those of expected intermediates resulting from partial protease cleavage of the three-cistron polyprotein. On the other hand, the only detectable HC-related product of the full-length clone was a mature-size HC. The presence of a third PVY protease among the first three cistrons is therefore suggested.

Expression of active human interferon-beta in transgenic plants.

Tobacco plants were transformed with the human gene for interferon-beta (IFN-beta). Transformation was determined by the polymerase chain reaction (PCR), and expression was determined by Western blot analysis, by purifying the IFN from the transgenic plants, and by bioassays indicating its activity in human cells. Plants expressing IFN-beta were self-pollinated. IFN-beta-expressing progeny plants were selected and produced active IFN-beta, indicating stable transformation.

Sheep bronchoalveolar carcinoma: tissue associated protein complex (TAPC) in normal lung tissue and in the tumor differed quantitatively.

Sheep lungs experimentally and naturally affected by bronchoalveolar carcinoma were washed out exhaustively of soluble components by phosphate-buffered saline, pH 7.4 (PBS), followed by glycine buffer, pH 2.8 (GB), and then again by 1M KCl followed by PBS. The tissue matrix (TM) of the tumor-free region and the tumor-affected tissue were analysed separately by sodium dodecyl sulfate (SDS) polyacrylamide electrophoresis. Normal lung tissues obtained from normal sheep served as controls. Several protein fractions and fragments, identified in both the normal and the tumorous lung, have the molecular weight (MW) of 130,000-228,000, as compared with the major soluble tissue associated protein having MW of 70,000. Coomassie blue staining used in the SDS polyacrylamide system and alkaline phosphatase immunoreaction used in the Enzyme Linked Immunosorbant Assay (ELISA) showed tenfold increased concentration of the TAPC in the TM of the tumor tissue and in the blood of tumor-affected animals, respectively. Total concentration of the TAPC in the serum of tumor-affected animals was higher than in the normal. Immunofluorescent antibody test (IAT) detected the TAPC in the cytoplasm of tumor as well as in normal lung cells, and the study suggested that the TAPC reaches the peripheral blood during tissue destruction occurring at the tumor site, as observed by light and electron microscopy (LM and EM). The concentration of each of the TAPC fractions was higher in the tumor-affected sheep lung as compared with normal sheep lung. Antibodies prepared against the TAPC fractions were toxic to sheep lung cells in tissue culture. Tumor cells were more susceptible.

The contribution of thermo-moxibustion to surgical treatment in transplanted mouse mammary carcinoma.

BALB/C mice bearing progressive transplanted mammary carcinoma were treated by surgical removal of the tumor mass either with or without thermo-moxibustion, applied to the GV 14-point-equivalent. A surgical removal of the tumor, 14 days post-inoculation resulted in 61% of deaths compared with 90.0% of deaths in the sham operated control. By the addition of the thermo-moxibustion therapy, the mortality rate, in the surgically treated group was reduced to 37.5%. Surgical removal of the tumor mass at day 17 post-inoculation resulted in 70% death while surgery supported by thermo-moxibustion protected the animals to the range of 40% death. Surgical removal of the tumor mass followed by local heterologous immuno-therapy was very effective when applied 14 days post-inoculation (12.5% death only). However, the same procedures applied at day 17 post-inoculation were not effective (80% death). Surgery and moxibustion together were more effective than surgery alone. Surgery and immunotherapy were very effective when applied from day 14 post-inoculation. Thermo-moxibustion as the sole treatment was effective when applied either before or very close to the tumor cell inoculation (35% of death and 33% of death respectively, as compared to 61.7% death in the control). Thermo-moxibustion applied before and after tumor cell inoculation was more effective (15% death rate). Thermo-moxibustion either alone or in conjunction with surgery is a contribution to the protection of BALB/C mice against the early and the late development of mouse mammary carcinoma.