Detection of early colorectal cancer imaged with peanut agglutinin-immobilized fluorescent nanospheres having surface poly(N-vinylacetamide) chains.

Peanut agglutinin (PNA)-immobilized fluorescent nanospheres were designed as a novel imaging agent for colonoscopy. PNA is a targeting moiety that binds to beta-D-galactosyl-(1-3)-N-acetyl-D-galactosamine, which is the terminal sugar of the Thomsen-Friedenreich antigen that is specifically expressed on the mucosal side of colorectal cancer cells. The in vivo performance of the imaging agent was evaluated using a human colorectal cancer orthotopic animal model. Human colorectal adenocarcinoma cell lines, HT-29, HCT-116, and LS174T, were implanted on the cecal serosa of immune-deficient mice. A loop of the tumor-bearing cecum was made, and the luminal side was treated with the imaging agent. Strong fluorescence was observed at several sites of the cecal mucosa, irrespective of cancer cell type. Microscopic histological evaluation of the cecal mucosa revealed that bright areas with fluorescence derived from the imaging agent and dark areas without the fluorescence well denoted the presence and absence, respectively, of the invasion of implanted cancer cells on the mucosal side. This good correlation showed that PNA-immobilized fluorescent nanospheres recognized millimeter-sized tumors on the cecal mucosa with high affinity and specificity.

In vitro/in vivo biorecognition of lectin-immobilized fluorescent nanospheres for human colorectal cancer cells.

Peanut agglutinin (PNA)-immobilized polystyrene nanospheres with surface poly(N-vinylacetamide) (PNVA) chains encapsulating coumarin 6 were designed as a novel colonoscopic imaging agent. PNA was a targeting moiety that binds to beta-D-galactosyl-(1-3)-N-acetyl-D-galactosamine, which is the terminal sugar of the Thomsen-Friedenreich antigen that is specifically expressed on the mucosal side of colorectal cancer cells. PNVA was immobilized with the aim of reducing nonspecific interactions between imaging agents and normal tissues. Coumarin 6 was encapsulated into nanosphere cores to provide endoscopically detectable fluorescence intensity. After incubation of imaging agents with human cells, the fluorescence intensity of imaging agent-bound cells was estimated quantitatively. The average fluorescence intensity of any type of colorectal cancer cell used in this study was higher than that of small intestinal epithelial cells that had not exposed the carbohydrate. The in vivo performance of imaging agents was subsequently evaluated using a human colorectal cancer orthotopic animal model. Imaging agent-derived strong fluorescence was observed at several sites of the large intestinal mucosa in the tumor-implanted nude mice after the luminal side of the colonic loop was contacted with imaging agents. In contrast, when mice that did not undergo tumor implantation were used, the fluorescence intensity on the mucosal surface was extremely low. Data indicated that imaging agents bound to colorectal cancer cells and the cancer cell-derived tumors with high affinity and specificity.

Structural characterisation and biological activities of a unique type beta-D-glucan obtained from Aureobasidium pullulans.

A beta-D-glucan obtained from Aureobasidium pullulans (AP-FBG) exhibits various biological activities: it exhibits antitumour and antiosteoporotic effects and prevents food allergies. An unambiguous structural characterisation of AP-FBG is still awaited. The biological effects of beta-D-glucan are known to depend on its primary structures, conformation, and molecular weight. Here, we elucidate the primary structure of AP-FBG by NMR spectroscopy, and evaluate its biological activities. Its structure was shown to comprise a mixture of a 1-3-beta-D-glucan backbone with single 1-6-beta-D-glucopyranosyl side-branching units every two residues (major structure) and a 1-3-beta-D-glucan backbone with single 1-6-beta-D-glucopyranosyl side-branching units every three residues (minor structure). Furthermore, this beta-D-glucan exhibited immunostimulatory effects such as the accumulation of immune cells and priming effects against enterobacterium. To our knowledge, 1-3-beta-glucans like AP-FBG with such a high number of 1-6-beta-glucopyranosyl side branching have a unique structure; nevertheless, many 1-3-beta-glucans were isolated from various sources, e.g. fungi, bacteria, and plants.

Poly(N-vinylacetamide) chains enhance lectin-induced biorecognition through the reduction of nonspecific interactions with nontargets.

Lectin-immobilized fluorescent nanospheres were designed with the aim of developing a novel endoscopic imaging agent for the detection of early colorectal cancer. Submicron-sized polystyrene nanospheres with surface poly(N-vinylacetamide) (PNVA) and poly(methacrylic acid) (PMAA) chains encapsulating fluorescein-labeled cholesterol were prepared as a platform of the imaging agent. Peanut agglutinin (PNA) was immobilized on the surface of fluorescent nanospheres through a chemical reaction with PMAA in order to recognize beta-D-galactosyl-(1-3)-N-acetyl-d-galactosamine (Gal-beta(1-3)GalNAc), which is the terminal sugar of the Thomsen-Friedenreich antigen that is specifically expressed on the mucosal side of colorectal cancer cells. The effect of surface structure of nanospheres on the affinity and specificity of immobilized PNA for Gal-beta(1-3)GalNAc was examined. Agglutination of normal and Gal-beta(1-3)GalNAc-expressed erythrocytes in the presence of nanospheres showed that PNA was immobilized actively on the nanosphere surface. Molecular weights of PNVA and PMAA affected the PNA activity most strongly. When the weight-average molecular weight of PNVA was nearly equal to that of PMAA, the affinity of PNA immobilized on the nanosphere surface for Gal-beta(1-3)GalNAc was as strong as that of intact PNA; the specificity for the carbohydrate residue was higher than that of the PNA. Results indicated that PNVA enhanced the specificity of PNA through the reduction of nonspecific interactions between PNA and carbohydrates other than Gal-beta(1-3)GalNAc on the erythrocyte surface without a significant decrease in the affinity.

Heterologous mevalonate production in Streptomyces lividans TK23.

Mevalonate is a ubiquitous biosynthetic intermediate of terpenoids and is used as a moisturizer in cosmetics and a chemical for biochemical research. In this study, we have achieved a heterologous production of this useful compound by expression in Streptomyces lividans TK23 of 3-hydroxy-3-methylglutaryl-CoA synthase and 3-hydroxy-3-methylglutaryl-CoA reductase genes, which were cloned from Streptomyces sp. strain CL190.