Growth stimulation of non-small cell lung cancer cell lines by antibody against epidermal growth factor receptor promoting formation of ErbB2/ErbB3 heterodimers

Antibodies are the most rapidly expanding class of human therapeutics, including their use in cancer therapy. Monoclonal antibodies (mAb) against epidermal growth factor (EGF) receptor (EGFR) generated for cancer therapy block the binding of ligand to various EGFR-expressing human cancer cell lines and abolish ligand-dependent cell proliferation. In this study, we show that our mAb against EGFRs, designated as B4G7, exhibited a growth-stimulatory effect on various human cancer cell lines including PC-14, a non-small cell lung cancer cell line; although EGF exerted no growth-stimulatory activity toward these cell lines. Tyrosine phosphorylation of EGFRs occurred after treatment of PC-14 cells with B4G7 mAb, and it was completely inhibited by AG1478, a specific inhibitor of EGFR tyrosine kinase. However, this inhibitor did not affect the B4G7-stimulated cell growth, indicating that the growth stimulation by B4G7 mAb seems to be independent of the activation of EGFR tyrosine kinase. Immunoprecipitation with anti-ErbB3 antibody revealed that B4G7, but not EGF, stimulated heterodimerization between ErbB2 and ErbB3. ErbB3 was tyrosine phosphorylated in the presence of B4G7 but not in the presence of EGF. Further, the phosphorylation and B4G7-induced increase in cell growth were inhibited by AG825, a specific inhibitor of ErbB2. These results show that the ErbB2/ErbB3 dimer functions to promote cell growth in B4G7-treated cells. Changes in receptor-receptor interactions between ErbB family members after inhibition of one of its members are of potential importance in optimizing current EGFR family-directed therapies for cancer.     

Novel DPPH radical scavengers, demethylbisorbibutenolide and trichopyrone, from a fungus

In our screening program for antioxidants with 1,1-diphenyl-2-picrylhydrazyl (DPPH)-radical scavenging activity, two novel compounds, demethylbisorbibutenolide (1) and trichopyrone (2), were isolated from the fermentation broth of the fungus of USF-4860 strain isolated from a soil sample. The structures of these compounds were determined from spectroscopic evidence. The biosynthetic origin of the carbon atoms of 2 was unambiguously determined by feeding experiments using (13)C-labeled precursors and elucidation of the (13)C-NMR spectrum of (13)C-labeled 2. These studies showed that 2 was derived from five acetates and a methyl group of methionine. In the DPPH-radical scavenging assay, 1 and 2 gave ED(50) values of 149 and 167 muM after standing for 2.0 hr. Compound 2 reacted with the DPPH radical to form reaction product 3 which was determined to be 1-[4-(3,4-dihydro-3-methyl-6-{1,3-pentadienyl}-2,4-dioxo-2H-pyran-3-yl)-phenyl]-1-phenyl-2-picrylhydrazine from spectroscopic evidence.     

Re-established mutualism in a seed-dispersal system consisting of native and introduced birds and plants on the Bonin Islands,Japan

The disruption of plant–animal interactions such as seed dispersal is one of the most critical effects of biological invasions. To understand the role of introduced species in current seed-dispersal systems, we conducted fecal analyses of the most common resident land birds on the Bonin Islands, Japan, and estimated their relative importance as seed-dispersal agents. Two native birds, the brown-eared bulbul and the Bonin Islands white-eye, and the introduced Japanese white-eye were the primary seed dispersers in secondary forest sites. Because the seed species composition in the feces of native and introduced white-eyes was similar, the latter may be replacing the former as a seed-dispersal agent. Introduced plants did not decrease the number of seed-dispersal opportunities for native species through competition for seed dispersers. Because some bird species have already become extinct on the Bonin Islands, their ecological functions may also have been permanently lost; however, the introduced white-eye may be compensating for this loss of function. In addition, new mutualistic relationships involving native and introduced birds and plants have already been established. In order to control introduced species while having the least impact on the native biota, interspecific interactions must be thoroughly understood before initiating control efforts.     

Design, synthesis and biological activity of novel non-peptidyl endothelin converting enzyme inhibitors, 1-phenyl-tetrazole-formazan analogues

A novel non-peptidyl endothelin converting enzyme inhibitor was obtained through a pharmacophore analysis of known inhibitors and three-dimensional structure database search. Analogues of the new inhibitor were designed using the structure-activity relationship of known inhibitors and synthesized. In anesthetized rats, intraperitoneal administration of the analogues suppressed the pressor responses induced by big endothelin-1.     

Oral-tolerance induction in diet-induced obese mice

OBJECTIVE: It is known that immune functions are altered in various ways by obesity. However, changes in the intestinal immune system resulting from obesity remain poorly understood. Oral tolerance is a system that suppresses antigen specific immune responses to orally administrated antigens. The intestinal immune system is intimately associated with the oral tolerance system, that acts to prevent allergic and inflammatory diseases. In this study we investigated the effect of obesity on induction of oral tolerance to ovalbumin (OVA) in an animal model of obesity. RESEARCH METHODS AND PROCEDURES: Obese mice induced by a high fat diet and control mice were allowed free access for 3 days to a 1%-ovalbumin (OVA) solution in drinking water. After continuous feeding of the antigen, all the mice were immunized by two intraperitoneal injections of OVA administered 7 days apart. RESULTS: In the control mice, induction of oral tolerance caused an increase in antigen specific IgG1 levels and a decrease in IgG2a levels. In contrast, the IgG1/IgG2a ratio was reversed in obese mice. OVA-specific IL-2 production was suppressed by antigen feeding in both the control and obese mice; however, suppression of OVA-specific IL-10 was observed only in the control mice. Although OVA-specific IgA and IgM were not affected by antigen feeding, the obese groups of mice had significantly lower titers of antibodies. DISCUSSION: These findings suggest that obesity may affect induction of oral tolerance following antigen feeding and that these changes may be related to the inflammatory reaction.     

A newly established GDL1 cell line from gpt delta mice well reflects the in vivo mutation spectra induced by mitomycin C

In order to create a novel in vitro test system for detection of large deletions and point mutations, we developed an immortalized cell line. A SV40 large T antigen expression unit was introduced into fibroblasts derived from gpt delta mouse lung tissue and a selected clone was established as the gpt delta L1 (GDL1) cell line. The novel GDL1 cells were examined for mutant frequencies (MFs) and for molecular characterization of mutations induced by mitomycin C (MMC). The GDL1 cells were treated with MMC at doses of 0.025, 0.05, and 0.1 microg/mL for 24h and mutations were detected by Spi- and 6-thioguanine (6-TG) selections. The MFs of the MMC-treated cells increased up to 3.4-fold with Spi- selection and 3.5-fold with 6-TG selection compared to MFs of untreated cells. In the Spi- mutants, the number of large (up to 76 kilo base pair (kbp)) deletion mutations increased. A majority of the large deletion mutations had 1-4 base pairs (bp) of microhomology in the deletion junctions. A number of the rearranged deletion mutations were accompanied with deletions and insertions of up to 1.1 kbp. In the gpt mutants obtained from 6-TG selection, single base substitutions of G:C to T:A, tandem base substitutions occurring at the 5'-GG-3' or 5'-CG-3' sequence, and deletion mutations larger than 2 bp were increased. We compared the spectrum of MMC-induced mutations observed in vitro to that of in vivo using gpt delta mice, which we reported previously. Although a slight difference was observed in MMC-induced mutation spectra between in vitro and in vivo, the mutations detected in vitro included all of the types of mutations observed in vivo. The present study demonstrates that the newly established GDL1 cell line is a useful tool to detect and analyze various mutations including large deletions in mammalian cells.     

High frequency of mutations at position 11778 in mitochondrial ND4 gene in Japanese families with Leber's hereditary optic neuropathy

We have investigated the presence of a point mutation at position 11778 in the ND4 gene of mitochondrial DNA in 17 Japanese families with Leber's hereditary optic neuropathy (LHON), and have identified the mutation in 14 (82.4%) of the 17 families. The prevalence of this mutation appears to be much higher in Japanese patients with LHON than in patients of other ethnic origins, such as Finnish, Dutch, German, and English families.     

Endothelin Receptors in Rat Pituitary Gland

1. We used the quantitative receptor autoradiographic method plus ¹²⁵I-endothelin-1 (¹²⁵I-ET-1), BQ-123, a specific antagonist for the endothelin ET_A receptor, and sarafotoxin S6c, a selective agonist for the ET_B receptor to investigate the ET receptor in the rat pituitary gland.2. The method revealed that the BQ-123-sensitive ET_A receptor was present predominantly in the anterior lobe and Rathke's pouch.3. The posterior lobe contained BQ-123-sensitive ET_A and sarafotoxin S6c-sensitive ET_B receptors, in almost the same proportion. There was no significant ¹²⁵I-ET-1 binding to the intermediate lobe.4. Knowledge of the heterogeneous distribution of ET receptor subtypes in the pituitary gland supplies information that will be pertinent to physiological investigations of the gland.