The polysaccharides, such as κ-carrageenan, ι-carrageenan, agarose (agar), gellan gum, amylose, curdlan, alginate, and deacetylated rhamsan gum, in water changed into an ice-like structure with hydrogen bonding between polymer and water molecules, and between water-water molecules even at a concentration range of 0.1%-1.0% (W/V) at room temperature, resulting in gelation. Such dramatic changes from liquid into gels have been understood at the molecular level in principles. In this review, we describe the structure-function relationship of starch on the view point of rheological aspects and discuss gelatinization and retrogradation mechanism including water molecules at molecular level. The starch molecules (amylose and amylopectin) play a dominant role in the center of the tetrahedral cavities occupied by water molecules, and the arrangement is partially similar to a tetrahedral structure in a gelatinization process. The arrangement should lead to a cooperative effect stabilizing extended regions of ice-like water with hydrogen bonding on the surface of the polymer molecules, where hemiacetal oxygen and hydroxyl groups might participate in hydrogen bonding with water molecules. Thus, a more extended ice-like hydrogen bonding within water molecules might be achieved in a retrogradation process. Though many investigations not only include starch gelatinization and retrogradaion, but also the gelling properties of the polysaccharides have been undertaken to elucidate the structure-function relationship, no other researchers have established mechanism at the molecular level. There is reasonable consistency in our investigations.

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We investigated a structural characteristics of acetyl fucoidan (CAF) isolated from commercially cultured Cladosiphon okamuranus. The CAF-induced macrophage activation and its signaling pathways in murine macrophage cell line, RAW 264.7 were also investigated. From the results of methylation analysis, CAF consisted of alpha-1-->3 linked L: -fucosyl residues and substituted sulfate and acetyl groups at C-4 on the main chain. CAF induced production of nitric oxide (NO), tumor necrosis factor-alpha and interleukin-6 in RAW 264.7 cells. Sulfate and acetyl groups of CAF involved in CAF-induced NO production. Neutralizing anti-Toll-like receptor 4 (TLR4), anti-CD14 and anti-scavenger receptor class A (SRA) but not anti-complement receptor type 3 monoclonal antibodies decreased CAF-induced NO production. The results of immunoblot analysis indicated that CAF activated mitogen-activated protein kinases (MAPKs) such as p38 MAPK, extracellular signal-regulated kinase (ERK)1/2 and stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). SB203580 (p38 MAPK inhibitor) and SP600125 (SAPK/JNK inhibitor), but not U0126 (MAPK/ERK kinase 1/2 inhibitor) decreased CAF-induced NO production. The results suggested that CAF induced macrophage activation through membrane receptors TLR4, CD14 and SRA, and MAPK signaling pathways.

A galactomannan isolated from seeds of Delonix regia was investigated. The specific rotation of the galactomannan was +15 degrees at 25 degrees C. The molecular mass of the galactomannnan was estimated to be 2.5 x 10(5). The ratio of D-mannose to D-galactose was estimated approximately to be 4.0:1.0 by HPLC and (1)H-NMR. NMR ((1)H and (13)C) spectra, and methylation analysis of the galactomannan indicated that it was composed of 1,4-linked beta-D-mannose, 1,4,6-linked beta-D-mannose, and terminal alpha-D-galactose. It was composed of pentasaccharide repeating-units, the chemical structure of which is proposed below.

Engineered T-cell therapy using a CD19-specific chimeric antigen receptor (CD19-CAR) is a promising strategy for the treatment of advanced B-cell malignancies. Gene transfer of CARs to T-cells has widely relied on retroviral vectors, but transposon-based gene transfer has recently emerged as a suitable nonviral method to mediate stable transgene expression. The advantages of transposon vectors compared with viral vectors include their simplicity and cost-effectiveness. We used the Tol2 transposon system to stably transfer CD19-CAR into human T-cells. Normal human peripheral blood lymphocytes were co-nucleofected with the Tol2 transposon donor plasmid carrying CD19-CAR and the transposase expression plasmid and were selectively propagated on NIH3T3 cells expressing human CD19. Expanded CD3+ T-cells with stable and high-level transgene expression (~95%) produced interferon-γ upon stimulation with CD19 and specifically lysed Raji cells, a CD19+ human B-cell lymphoma cell line. Adoptive transfer of these T-cells suppressed tumor progression in Raji tumor-bearing Rag2−/−γc−/− immunodeficient mice compared with control mice. These results demonstrate that the Tol2 transposon system could be used to express CD19-CAR in genetically engineered T-cells for the treatment of refractory B-cell malignancies.

We investigated macrophage activation by fucoidan from Laminaria angustata var. longissima in a murine macrophage cell line, RAW 264.7. The ratio of the chemical composition of the fucoidan was L-fucose: D-galactose:D-glucose:D-xylose:uronic acid:sulfate = 1.00:0.54:0.08:0.08:0.64:0.87. The fucoidan induced production of nitric oxide, tumor necrosis factor-, and interleukin-6 in RAW 264.7 cells. These results indicate that the fucoidan induced macrophage activation.Key words: Laminaria angustata var. longissima; fucoidan; macrophage activationFucoidan is a sulfated polysaccharide found in the cell-wall matrix of brown algae. There have been many reports that fucoidans from various brown algae have biological activities, such as anti-tumor, 1) anticoagulant, 2) anti-human immuno-deficiency virus, 3) and induction of apoptosis. 4) We have isolated a novel acetyl fucoidan from Cladosiphon okamuranus, 5,6) and have reported macrophage activation by acetyl fucoidan. 7)Macrophages are multifunctional cells that play significant roles in host defense. They phagocyte an invaded pathogen and produce inflammatory mediators and cytokines. Nitric oxide (NO), which is known to be an inflammatory mediator, is produced by the activation of inducible nitric oxide synthase (iNOS) from Larginine.8) NO has been reported to play an essential role in the cytolytic function, the killing of pathogens, and the tumoricidal action.9-11) Cytokines have an important role in the regulation of the inflammatory and immune responses.12) Polysaccharides isolated from plants and algae have been reported to enhance macrophage activation. 13-15)Laminaria angustata var. longissima is a brown alga eaten widely since 300 years ago in Okinawa, Japan. We have reported structural characteristics of fucoidans from L. angustata var. longissima.16) The fucoidans were obtained at about 1.4% w/w on the basis of dry matter, and they consisted of L-fucose, D-galactose, D-xylose, D-glucuronic acid, and sulfuric acid. We here investigated macrophage activation by the fucoidan in a murine macrophage cell line, RAW 264.7.Dulbecco's modified Eagle medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Gibco BRL (Grand Island, NY). AntiiNOS rabbit IgG monoclonal antibody and anti--actin rabbit IgG polyclonal antibody were from Sigma (St. Louis, MO).The total carbohydrate and uronic acid of the fucoidan was analyzed by the phenol-sulfuric acid method 17) and the carbazole-sulfuric acid method 18) respectively. The sulfate content of the fucoidan was measured by the turbidimetric method. 19)The monosaccharides in the hydrolysate of the fucoidan were determined using high-performance anion exchange chromatography (DX-500, Dionex, Sunnyvale, CA), and fitted with a column of Carbopack PA1 and a pulsed amperometric detector. The column was eluted at a flow rate of 1 ml/min at 35 C with 15 mM NaOH for neutral sugar.The molecular mass of the fucoidan was measured by high-performance liquid chromatography (LC-6A; Shimadzu, Kyoto, Japan) wit...

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