Yuko Setoguchi

Elevated expression levels of the 14-3-3 family of proteins in lung cancer tissues

Immunochemical staining of lung cancer sections with a murine monoclonal anti-14-3-3 antibody showed a sharp discrimination of the cancer tissue from neighboring normal counterparts in 88 of 121 primary lung cancer tissue specimens of all four major lung cancer histologies; specifically, 32 of 48 adenocarcinomas, 36 of 44 squamous cell carcinomas, 10 of 13 large cell carcinomas, and 10 of 16 small cell carcinomas, respectively, were stained positively. Sets of the 10,000 x g supernatants of normal and cancerous lung tissue homogenates, each set prepared from surgically dissected tissues of the cancer and its surrounding normal part, were assayed for 14-3-3 proteins by the sandwich enzyme-linked immunosorbent assay using two different monoclonal antibodies to 14-3-3 proteins. The results of the assay demonstrated 7.2 times higher 14-3-3 protein content in the lung cancer tissue (378 +/- 200 ng ml-1) as compared with the normal lung (54 +/- 35 ng ml-1). These results indicate that the 14-3-3 family of proteins can be an effective marker for lung cancer diagnosis such as sputum cytodiagnosis and that 14-3-3 proteins might be involved in the development of lung cancers.

Molecular cloning of the 31 kDa cytosolic phospholipase A2, as an antigen recognized by the lung cancer-specific human monoclonal antibody, AE6F4

The human monoclonal antibody AE6F4 specifically reacts with human lung cancer tissues but does not with normal tissues. This monoclonal antibody recognizes a cytosolic 31 kDa antigen in the cancer cells. In a previous study, we elucidated that the 31 kDa antigen belonged to a family of proteins collectively designated as 14-3-3 proteins, which were known as protein kinase-dependent activators of tyrosine/trytophan hydroxylases, or protein kinase C inhibitor proteins. Here we report molecular cloning of the 31 kDa antigen from the human lung adenocarcinoma cell line, A549. Sequencing analysis indicates that the cloned cDNA is identical to that of previously reported human placental cytosolic phospholipase A2 (cPLA2), which is also a member of the 14-3-3 protein family. Western analysis demonstrated that a 31 kDa recombinant cPLA2 expressed in monkey COS cells was recognized by the AE6F4 monoclonal antibody. Binding of the monoclonal antibody to the recombinant cPLA2 was abolished when treated with sodium periodate, suggesting that not only are carbohydrate chains associated with the cPLA2, but they also play a crucial role in antigen recognition by the monoclonal antibody.

Immunocytochemical detection of lung cancer cells with monoclonal antibodies to 14-3-3 proteins.

Murine monoclonal antibodies were raised against 14-3-3 proteins, the antigen of human monoclonal antibody AE6F4 which had been shown potentially useful for the immunochemical diagnosis of lung cancer via sputum cytology. Enzyme-linked immunosorbent assays of the murine anti-14-3-3 monoclonal antibodies with isolated bovine brain 14-3-3 isoforms showed that the antibodies were classified into four different profiles of isoform reactivity. The comparison of 14-3-3 isoform and lung cancer tissue on the reactivity with murine monoclonal antibodies indicated that beta isoform can be responsible for cancer recognition, whereas human monoclonal antibody AE6F4 showed preferential binding to zeta isoform. No murine monoclonal antibody of the same isoform specificity as human monoclonal antibody AE6F4 was obtained. Since murine monoclonal antibodies with different isoform specificities could immunostain lung cancer cells in sputum successfully, the combination use of murine monoclonal anti-14-3-3 antibodies with human monoclonal antibody AE6F4 is potentially useful for facilitating the sputum cytodiagnosis of lung cancer.

Lung cancer-reacting human recombinant antibody AE6F4: Potential usefulness in the sputum cytodiagnosis

Human monoclonal antibody (hMAb) AE6F4 has been shown to be potentially useful for immunocytological detection of lung cancer cells in sputum. By recombinant DNA technology, IgM type hMAb AE6F4 was switched to lgG. The IgG mimic recombinant AE6F4 antibody expression plasmid was assembled using the antibody heavy chain gene, which ligated the gene encoding VH and CH1(mu) domains of hMAb AE6F4 heavy chain to the gene encoding CH2(gamma 1) and CH3(gamma 1) domains of human IgG heavy chain, and the antibody light chain gene of hMAb AE6F4. The recombinant antibody expressed by baby hamster kidney (BHK)-21 cells showed molecular size equivalence to IgG, and consisted of human mu-gamma hybrid heavy and kappa light chains. The immunological specificity of the recombinant antibody was the same as that of hMAb AE6F4 by immunoblotting analysis to the 14-3-3 protein, the putative antigen of hMAb AE6F4, and by immunohistochemical and immunocytological analyses using tissue sections and sputa of lung cancer patients. The transfected BHK-21 cells produced the recombinant antibody persistently and the productivity was greater than 20 times that by human-human hybridoma producing hMAb AE6F4.

Cancer-specific binding of a mouse MAb vs. Candida krusei cytochrome c: an antigen recognized by a cancer-associated human MAb HB4C5.

More than 60 mouse monoclonal antibodies directed to cytochrome c from Candida krusei with different specificities were raised. Most of these monoclonal antibodies, except for three of them, did not cross-react with bovine cytochrome c. By the immunoblotting method, the monoclonal antibodies of clones HCC 5-13, 9-2, and 10-5 reacted with the Candida cytochrome c, which had been transferred onto nitrocellulose membrane, but those of clones HCC 1-22, 6-3, and 17-3 did not, although all these monoclonal antibodies strongly reacted with coated Candida cytochrome c on plastic immunoplates when examined by ELISA. On the contrary, monoclonal antibody activities of clones HCC 1-22, 6-3, and 17-3 in binding to the coated cytochrome c in ELISA were inhibited competitively by the addition of extra Candida cytochrome c, whereas those of clones HCC 5-13, 9-2, and 10-5 were not inhibited. Among these monoclonal antibodies, the antibody of clone HCC 6-3, which showed a good reactivity to added cytochrome c in inhibiting ELISA reaction but was not reactive with the transblotted cytochrome c on nitrocellulose, was found to be reactive with human lung cancer tissues specifically with no reactivity to normal tissues. The immunostaining of lung cancer tissue showed that this mouse monoclonal antibody to Candida cytochrome c reacted to the cytoplasmic fraction of the cancer cells specifically.

高脂肪飼料摂取マウスにおける「べにふうき」緑茶の脂肪蓄積抑制効果

benifuuki, methylated catechins, epigallocatechin-( -methyl)gallate (EGCG Me), fat accumulation, obesity : : EGCG Me Anti-obese e ects of tea leaf powders were compared in mice who consumed ‘benifuuki’, a tea cultivar that contains unique methylated catechins such as epigallocatechin-( -methyl) gallate (EGCG Me), and ‘yabukita’, a popular tea cultivar in Japan that lacks methylated catechins. For weeks, four groups of -week-old male C BL/ J mice ( per group) were fed either high-fat diets with and without the addition of benifuuki or yabukita tea leaf powder ; for a non-obese control group, mice were fed a low-fat diet with no additive. The body weight and food intake were recorded for each mouse times a week. On the last day of the experiment, the mice were sacrificed to determine the final weight of white adipose tissue as well as blood levels of lipid metabolism-related components. The hot-water extract of benifuuki, an ingredient useful for industrial applications, was similarly examined in mice for anti-obese e ects and dose dependency by a single daily oral administration of , and mg total catechins per kg body weight. The mice fed a high-fat diet containing benifuuki powder showed significant decreases in body weight, adipose tissue weights and plasma leptin concentration, compared with those fed a control high-fat diet with no additive. On the other hand, the mice fed a high-fat diet containing yabukita powder showed less anti-obese e ects, with the only significance in subcutaneous fat weight. A single daily administration of benifuuki extract to mice that were fed a high-fat diet showed anti-obese e ects in a dosedependent manner. The results indicated that benifuuki had much higher anti-obese e ects than yabukita, and was dose dependent. The strong anti-obese activity of benifuuki may be ascribed to the unique methylated catechins (primarily EGCG Me) contained exclusively in this cultivar, whose fractional absorption and stability in blood are much higher than EGCG. Health Food Science Research Institute, Morinaga & Co., Ltd., , Shimosueyoshi, Tsurumi-ku, Yokohama, Kanagawa * Morinaga Institute of Biological Science, INC., , Sachiura, Kanazawa-ku, Yokohama, Kanagawa ** National Institute of Vegetable and Tea Science, National Agriculture and Food Research Organization, Kanaya, Shimada, Shizuoka

Administration of Piceatannol Complexed with α-Cyclodextrin Improves Its Absorption in Rats

Piceatannol is polyphenolic antioxidant found in passion fruit (Passiflora edulis) seeds. The aim of this study was to improve the absorption of piceatannol using α-cyclodextrin (αCD). The solubility of piceatannol in neutral and acidic solutions increased in an αCD concentration-dependent manner. The maximum plasma concentration of intact piceatannol and the time-to-maximum plasma concentration of O-methylated piceatannol metabolites increased in rats administered αCD-piceatannol inclusion complexes (PICs). Administering the αCD inclusion complexes significantly increased the area under the concentration-time curve of total stilbene derivatives (0-3 h) in terms of the total amount of intact piceatannol, O-methylated piceatannol, conjugated piceatannol, and isorhapontigenin. Gastrointestinal ligation experiments demonstrated that substantially higher levels of piceatannol metabolites were present in the lower intestine (the ileum) at 1 h postintragastric αCD-PICs administration as compared to those observed following piceatannol administration only. These results suggested that αCD enhanced piceatannol movement and absorption in the small intestine.

The 31 KD Cytosolic Phospholipase A2 is a Target Molecule for the Cancer-Specific Human Monoclonal Antibody, AE6F4

The 31 kDa cytosolic phospholipase A2 (cPLA2) has been shown to be a target molecule for the cancer-specific human monoclonal antibody, AE6F4. The 31 kDa cPLA2 is a member of a family of proteins collectively designated as 14–3–3 proteins, which are known as protein kinase-dependent activators of tyrosine/tryptophan hydroxylases, and protein kinase C inhibitor proteins. Here, we cloned the cPLA2 cDNA from the human lung adenocarcinoma line A549, and introduced this cDNA into COS cells to express recombinant cPLA2. We show that the recombinant cPLA2 is recognizable by the AE6F4 MAb. Northern blot analysis revealed that the mRNA of several 14–3–3 protein family members are seen in A549 cells.