Importance of co-cultivation medium pH for successful Agrobacterium-mediated transformation of Lilium x formolongi.

An efficient system for Agrobacterium-mediated transformation of Lilium x formolongi was established by preventing the drastic drop of pH in the co-cultivation medium with MES. Meristematic nodular calli were inoculated with an overnight culture of A. tumefaciens strain EHA101 containing the plasmid pIG121-Hm which harbored intron-containing beta-glucuronidase (GUS), hygromycin phosphotransferase (HPT), and neomycin phosphotransfease II (NPTII) genes. After three days of co-cultivation on 2 g/l gellan gum-solidified MS medium containing 100 microM acetosyringone, 30 g/l sucrose, 1 mg/l picloram and different concentrations of MES, they were cultured on the same medium containing 12.5 mg/l meropenem to eliminate Agrobacterium for 2 weeks and then transferred onto medium containing the same concentration of meropenem and 25 mg/l hygromycin for selecting putative transgenic calli. Transient GUS expression was only observed by adding MES to co-cultivation medium. Hygromycin-resistant transgenic calli were obtained only when MES was added to the co-cultivation medium especially at 10 mM. The hygromycin-resistant calli were successfully regenerated into plantlets after transferring onto picloram-free medium. Transformation of plants was confirmed by histochemical GUS assay, PCR analysis and Southern blot analysis.

Expression of CD80 and CD86 on peripheral blood T lymphocytes in patients with systemic lupus erythematosus.

CD80 and CD86 were detected in high amounts on circulating T cells in the peripheral blood of some patients with systemic lupus erythematosus (SLE), using flow cytometry and monoclonal antibodies. Patients with other connective tissue diseases did not have a high percentage of T cells expressing CD80 or CD86 in their peripheral blood. CD80 was expressed mainly on CD4 T cells, whereas CD86 was expressed on CD8 T cells, and these two populations were associated with particular clinical features. These two molecules were expressed on different T-cell populations and might have different roles in the generation and regulation of immune responses. Since high expression of CD86 on T cells was detected much earlier than the appearance of clinical features and a high titer of anti-DNA antibody, it may be a useful parameter for predicting the flare of SLE.

Expression of costimulatory molecule CD80 on peripheral blood T cells in patients with systemic lupus erythematosus.

OBJECTIVE: To investigate the expression of costimulatory molecule CD80 on T cells of peripheral blood mononuclear cells (PBMC) in patients with systemic lupus erythematosus (SLE). METHODS: Monoclonal antibodies against CD80 were used for flow cytometry and expression of CD80 on PBMC was studied in 26 patients with SLE, 18 patients with rheumatoid arthritis (RA), 8 patients with mixed connective tissue disease (MCTD), and 22 healthy controls. RESULTS: CD80 was detected on CD3+ and CD19+ cells in patients with SLE and it was significantly higher than that of controls. In patients with SLE CD80 was expressed on CD4+ T cells (8.05+/-5.45%), significantly higher than in RA and controls, but was not highly expressed on CD8+ T cells (1.67+/-2.87%). CD80+CD4+ T cell phenotype analysis revealed CD45RA-, CD45RO+, and CD25+, or HLA-DR+ activated T cells. The percentage of CD80+ cells in CD4+ cells increased in the active stage of SLE, and was significantly correlated with the SLE disease activity index. CONCLUSION: CD80 can be expressed on activated CD4+ T cells in PBMC of patients with SLE in vivo and the appearance of these cells is associated with the disease activity in SLE.

Overexpression of bax sensitizes breast cancer MCF-7 cells to cisplatin and etoposide.

Bax, one of the bcl-2 family genes, is expressed in a number of untransformed cell lines and various breast tissues, whereas only weak or no expression has been detected in breast cancer cell lines and malignant breast tissue. Human breast cancer MCF-7 cells, which have a weak bax gene expression, were stably transfected with pCX2neo bax, encoding human bax; and two unique clones, MCF-7/bax-1 and MCF-7/ bax-2, that expressed different levels of bax were generated. Sensitivity to cisplatin (CDDP) and etoposide (VP-16) was examined and each stable transfectant was more sensitive to these agents than the parental MCF-7 cells. The degree of enhancement in sensitivity to these anticancer agents was dependent on the expression level of bax. The enzyme-linked immunosorbent assay (ELISA), which quantifies DNA damage, demonstrated that this sensitization was due to apoptosis. Thus, we suggest that exogenous bax-alpha overexpression may be one of the factors determining cellular chemosensitivity in MCF-7 breast cancer cells and that it could be applied therapeutically to enhance chemosensitivity in breast cancer cells.

Increased apoptosis rate by hyperthermochemoradiotherapy for advanced rectal cancers.

Apoptosis induced in cancer cells by ionizing radiation, hyperthermia, and 5-fluorouracil (5-FU), termed "hyperthermochemoradiotherapy" (HCR), has been well studied in vitro; however, the role of apoptosis in the tumocidal effect of HCR for primary rectal cancers has not yet been clarified. Therefore, we examined the relationship between the therapeutic effect and induction rate of histological apoptosis in 16 patients with rectal cancers after HCR. Numerous Tunel-positive apoptotic cells were found in the tumor tissue after HCR, but few were found in the tumors which had not received HCR. The histological therapeutic effect was closely correlated to the rate of apoptosis. Thus, we suggest that HCR induces a therapeutic effect mainly through apoptosis in human rectal cancers.

Inhibition of colon cancer cell proliferation by antisense oligonucleotides targeting the messenger RNA of the Ki-ras gene.

Point mutations that activate the Ki-ras proto-oncogene are present in approximately 50% of human colorectal tumors and the activated Ki-ras gene is considered to play an important role in colorectal cancer cell proliferation. Five different colon cancer cell lines and two kinds of control cell lines were treated with antisense oligonucleotides complementary to the messenger RNA of Ki-ras. Treatment with antisense oligonucleotides at concentrations between 10 and 40 microM significantly and dose-dependently inhibited cell growth, colony formation and Ki-ras protein production of the colon cancer cells with activated Ki-ras, but did not affect the normal cells and colon cancer cells without Ki-ras mutation. These results show that use of synthetic oligonucleotides is an effective way of producing antisense-mediated changes in the behavior of human colon cancer cells with an activated Ki-ras gene.

The anti-proliferative effect of proliferating cell nuclear antigen-specific antisense oligonucleotides on human gastric cancer cell lines.

The proliferating cell nuclear antigen (PCNA) is a nuclear protein that leads DNA synthesis by the DNA polymerase delta. As the PCNA gene is strongly expressed in invasive gastric cancer cells with high proliferative activity, PCNA is suspected of playing an important role in the proliferation and advancement of gastric cancer. Thus, the effects of antisense oligonucleotides specific for PCNA mRNA were examined in seven gastric cancer cell lines. It was found that treatment with antisense oligonucleotides at concentrations of 10-40 microM dose-dependently inhibited the growth of all cell lines; however, random sequence oligonucleotides did not modify the proliferation of any type of cells. These results indicate that PCNA is essential for cell proliferation in gastric cancer cells, and that the growth inhibitory effect results from the inhibition of PCNA gene expression. Therefore, PCNA-specific antisense oligonucleotides may be effective in the treatment of gastric cancer.

Inhibition of gastric cancer cell proliferation by antisense oligonucleotides targeting the messenger RNA encoding proliferating cell nuclear antigen.

Proliferating cell nuclear antigen (PCNA) is a nuclear protein that regulates DNA synthesis by DNA polymerase delta, and is essential for DNA replication. PCNA expression level is related to the malignancy of gastric cancer cells. Seven different gastric cancer cell lines and two kinds of control cell lines were treated with antisense oligonucleotides complementary to the messenger RNA of PCNA. Treatment of each gastric cancer cell line with antisense oligonucleotides at concentration of 10-40 microM inhibited the cell growth, colony formation and PCNA protein production in a dose-dependent manner, but only affected normal cells slightly. A random sequence oligomer showed no effect. These results show that PCNA is essential for gastric cancer cell proliferation and that the use of synthetic oligonucleotides is an effective way of producing antisense-mediated changes in the behaviour of human gastric cancers.

Evaluation of radioprotective action of a mutant (E-25) form of Chlorella vulgaris in mice.

The possible role of orally fed Chlorella vulgaris (E-25) in modulating the gamma-ray induced chromosomal damage in whole-body irradiated mice was evaluated using a micronucleus test. Different doses of E-25 were administered either chronically (once, twice or thrice a day for 28 days) or as single acute doses before/after irradiation. A significant radioprotective effect was observed in both acute and chronic pretreatments, but only at doses above 400 mg/kg body weight. However, in mice that received E-25 (500 mg/kg) three times a day for 28 days, there was no protective effect, and a significant loss in their body weight was observed. Interestingly, E-25 afforded significant radioprotection even when it was administered within 0.4 hr after irradiation.

Alteration in molecular species of phosphatidylethanolamine between anesthetic resistant and sensitive strains of Drosophila melanogaster.

An analysis of lipid composition was carried out in resistant and sensitive strains of Drosophila melanogaster. Amount of total lipid and amount of phosphate of phospholipids were not different from each other in both strains. Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) did not differ in amount between both strains. Determination of molecular species of PE using gas chromatography (GC) and GC-mass spectrometry showed that the resistant strain had increased 34:2 and decreased 36:2, 36:3 and 36:5 relative to the sensitive strain. The molecular species of PC did not differ between the two strains. Chromosomal analysis revealed that the alterations in 34:2 and 36:2 of PE were regulated by the X- and third chromosomes of the resistant strain. Therefore, the changes in PE may explain the mechanism of anesthetic resistance because genetic analyses indicate that these chromosomes have more influence on the anesthetic resistant traits of the resistant strain.

Strain differences in minimum anesthetic concentrations in Drosophila melanogaster.

An ether-resistant strain of Drosophila melanogaster has been maintained at this laboratory since the appearance of one female mutant in 1961. Sensitivity was defined using mortality as an endpoint when exposed to a high concentration of diethylether; this does not necessarily mean that anesthetic requirements are higher in the resistant strain. The present study was undertaken to determine the difference in anesthetic potency between the ether-resistant strain (Eth-29) and one of the sensitive strains (bw;st;svn). The median effective dose (ED50) for halothane was 0.0096 atm in females and 0.0091 atm in males of the bw;st;svn strain, while in the Eth-29 strain the ED50 was 0.0148 atm in both sexes. The ED50 values for chloroform anesthesia were 0.0051 atm in females and 0.0050 atm in males of the bw;st;svn strain and 0.0100 atm in the Eth-29 strain in both sexes. Strain differences in response to the two anesthetics were statistically significant. Thus the Eth-29 strain shows a cross-resistance to both halothane and chloroform anesthesia. Reciprocal crosses between the two strains revealed that the resistance to halothane anesthesia was a sex-linked recessive trait and that the resistance to chloroform anesthesia was an autosomal incompletely dominant trait.